T-cell receptor (tcr)-binding antibodies and uses thereof

ABSTRACT

Antibodies and antigen binding fragments thereof are provided that bind to T-cell receptors (e.g., TCRα), essentially independent of T-cell epitope specificity. Methods for manipulation of T-cells and methods of treatment using such antibodies are likewise provided.

This application is a continuation of U.S. application Ser. No.15/759,148, filed Mar. 9, 2018, as a national phase application under 35U.S.C. § 371 of International Application No. PCT/US2016/051847, filedSep. 15, 2016, which claims the benefit of U.S. Provisional ApplicationNo. 62/218,990, filed on Sep. 15, 2015, the entirety of each of which isincorporated herein by reference.

INCORPORATION OF SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 30, 2020, isnamed UTSCP1281USC1_ST25.txt and is 25 kb in size.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates generally to the field of molecularbiology and immunology. More particularly, it concerns monoclonalrecombinant antibodies that bind αβ-TCR (T-cell receptors) and the useof such antibodies in the manipulation of αβ-TCR⁺ T-cells inside andoutside the body.

Description of Related Art

T-cell based therapies are currently being explored for treatment of awide range of diseases. However, one of the major hurdles in anyT-cell-based therapy is efficient selection (isolation) and numericexpansion of T-cells for use in the therapy. Furthermore, T cells can bealtered inside the body for activation and immune suppression. Thus,there remains a need for new methods and compositions that can be usedfor manipulation of T-cells in vivo and ex vivo.

SUMMARY OF THE INVENTION

The invention provides an isolated antibody or antigen-binding fragmentthereof that specifically binds to an epitope of T-cell receptor alpha(TCRα) polypeptide comprising sequence GSTLRG (SEQ ID NO:1).

In embodiments, the antibody or antigen binding fragment thereofspecifically binds to an epitope of T-cell receptor alpha (TCRα)polypeptide comprising the sequence GSTLRG (SEQ ID NO:1) with anaffinity characterized by a dissociation constant (K_(D)) no greaterthan 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M,10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M,10⁻⁹ M, 5×10¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M,8.4×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴M, 5×10⁻¹⁵ M,or 10⁻¹⁵ M. In embodiments, the K_(D) is about 5×10⁻⁹M to about 6×10⁻⁹M. In further embodiments, the K_(D) is about 1×10⁻⁹M to about 2×10⁻⁹M.

In further embodiments, the invention relates to an antibody orantigen-binding fragment thereof that specifically binds to TCRαpolypeptide, comprising (a) HCDR1 at least 80%, 85%, 90% or 95%identical to SEQ ID NO: 2; (b) HCDR2 at least 80%, 85%, 90% or 95%identical to SEQ ID NO: 3; (c) HCDR3 identical to CDYW (SEQ ID NO:21);CAYW (SEQ ID NO:23); or CAYL (SEQ ID NO:22); (d) LCDR1 at least 80%,85%, 90% or 95% identical to SEQ ID NO: 4 or SEQ ID NO: 9; (e) LCDR2 atleast 80%, 85%, 90% or 95% identical to SEQ ID NO: 5; and (f) LCDR3 atleast 80%, 85%, 90% or 95% identical to SEQ ID NO: 6 or SEQ ID NO: 10.

The invention further provides an antibody or antigen-binding fragmentthereof that specifically binds to TCRα polypeptide comprising (a) HCDR1at least 80%, 85%, 90% or 95% identical to V_(H) CDR1 of 79A-15 SEQ IDNO: 2; (b) HCDR2 at least 80%, 85%, 90% or 95% identical to V_(H) CDR2of 79A-15 SEQ ID NO: 3; (c) HCDR3 at least 80%, 85%, 90% or 95%identical to V_(H) CDR3 of 79A-15 (SEQ ID NO:22); (d) LCDR1 at least80%, 85%, 90% or 95% identical to V_(L) CDR1 of 79A-15 SEQ ID NO: 9; (e)LCDR2 at least 80%, 85%, 90% or 95% identical to V_(L) CDR2 of 79A-15SEQ ID NO: 5; and (f) LCDR3 at least 80%, 85%, 90% or 95% identical toV_(L) CDR3 of 79A-15 SEQ ID NO: 10.

In further aspects, the invention provides an isolated antibody orantigen binding fragment thereof that specifically binds to TCRαpolypeptide, comprising (a) HCDR1 at least 80%, 85%, 90% or 95%identical to V_(H) CDR1 of 79A-13 KASGYTFTDYYMNWV (SEQ ID NO: 2); (b)HCDR2 at least 80%, 85%, 90% or 95% identical to V_(H) CDR2 of 79A-13WIGEINPNN (SEQ ID NO: 3); (c) HCDR3 at least 80%, 85%, 90% or 95%identical to V_(H) CDR3 of 79A-13 CAYL (SEQ ID NO:22); (d) LCDR1 atleast 80%, 85%, 90% or 95% identical to V_(L) CDR1 of 79A-13 NTYLEWY(SEQ ID NO: 4); (e) LCDR2 at least 80%, 85%, 90% or 95% identical toV_(L) CDR2 of 79A-13 KLLIYKVSNRFS (SEQ ID NO: 5); and (f) LCDR3 at least80%, 85%, 90% or 95% identical to V_(L) CDR3 of 79A-13 MQGSHVPW (SEQ IDNO: 10).

In embodiments, the invention further relates to an antibody orantigen-binding fragment thereof that specifically binds to TCRαpolypeptide, comprising (a) HCDR1 at least 80%, 85%, 90% or 95%identical to V_(H) CDR1 of 79A-11 KASGYTFTDYYMNWV (SEQ ID NO: 2); (b)HCDR2 at least 80%, 85%, 90% or 95% identical to V_(H) CDR2 of 79A-11WIGEINPNN (SEQ ID NO: 3); (c) HCDR3 at least 80%, 85%, 90% or 95%identical to V_(H) CDR3 of 79A-11 CAYW (SEQ ID NO:23); (d) LCDR1 atleast 80%, 85%, 90% or 95% identical to V_(L) CDR1 of 79A-11 NTYLEWF(SEQ ID NO: 9); (e) LCDR2 at least 80%, 85%, 90% or 95% identical toV_(L) CDR2 of 79A-11 KLLIYKVSNRFS (SEQ ID NO: 5); and (f) LCDR3 at least80%, 85%, 90% or 95% identical to V_(L) CDR3 of 79A-11 MQGSHVPW (SEQ IDNO: 10).

The invention also provides an isolated antibody or antigen bindingfragment thereof that specifically binds to TCRα polypeptide, comprising(a) HCDR1 at least 80%, 85%, 90% or 95% identical to V_(H) CDR1 of 79AKASGYTFTDYYMNWV (SEQ ID NO: 2); (b) HCDR2 at least 80%, 85%, 90% or 95%identical to V_(H) CDR2 of 79A WIGEINPNN (SEQ ID NO: 3); (c) HCDR3 atleast 80%, 85%, 90% or 95% identical to V_(H) CDR3 of 79A CDYW (SEQ IDNO:21); (d) LCDR1 at least 80%, 85%, 90% or 95% identical to V_(L) CDR1of 79A NTYLEWY (SEQ ID NO: 4); (e) LCDR2 at least 80%, 85%, 90% or 95%identical to V_(L) CDR2 of 79A KLLIYKVSNRFS (SEQ ID NO: 5); and (f)LCDR3 at least 80%, 85%, 90% or 95% identical to V_(L) CDR3 of 79AFQGSHVPW (SEQ ID NO: 6).

In embodiments, the invention provides an isolated antibody or antigenbinding fragment thereof that specifically binds to TCRα polypeptide,comprising (a) HCDR1 at least 80%, 85%, 90% or 95% identical toKASGYTFTGYYMNWV (SEQ ID NO:15); (b) HCDR2 at least 80%, 85%, 90% or 95%identical to WIGGINPNN (SEQ ID NO:16); (c) HCDR3 identical to CRYW (SEQID NO:17); (d) LCDR1 at least 80%, 85%, 90% or 95% identical toQSIVHGGGNTY (SEQ ID NO:18); (e) LCDR2 at least 80%, 85%, 90% or 95%identical to KLLIYKVSNRFS (SEQ ID NO: 5); and (f) LCDR3 at least 80%,85%, 90% or 95% identical to FQGSHVPW (SEQ ID NO: 6).

In additional aspects, the invention further provides an isolatedpolynucleotide comprising a nucleic acid encoding an antibody or antigenbinding fragment thereof that specifically binds to a T-cell Receptor achain (TCRα), said antibody binding to an epitope of TCRα polypeptidecomprising the sequence GSTLRG (SEQ ID NO: 1).

In further embodiments, the invention provides a vector comprising thepolynucleotides encoding an antibody or antigen binding fragment thereofthat specifically binds to a T-cell Receptor a chain (TCRα), saidantibody binding to an epitope of TCRα polypeptide comprising thesequence GSTLRG (SEQ ID NO: 1).

In still further embodiments, the invention provides a method ofmanufacturing an antibody or antigen binding fragment thereofcomprising: (a) expressing one or more polynucleotide molecule(s)encoding a V_(L) and V_(H) chain of an antibody in a cell; and (b)purifying the antibody from the cell, wherein the antibody specificallybinds to a T-cell Receptor a chain (TCRα).

The invention further provides a method for selecting a cell comprisinga T-cell Receptor a chain (TCRα) comprising: (a) contacting the cellwith an antibody or antigen fragment thereof that binds to a TCRα chain,wherein the antibody or fragment binds to T-cells having a plurality ofT-cell epitope specificities; and (b) selecting a cell comprising theTCR a chain based on binding of the antibody or fragment.

In additional embodiments, the invention provides a method for expandingand/or activating T-cells comprising contacting the T-cells withartificial antigen presenting cells (aAPCs) in the presence of anantibody or antigen binding fragment thereof that binds to an epitope ofa T-cell Receptor (TCR), wherein said epitope is a polypeptidecomprising sequence GSTLRG (SEQ ID NO:1).

In certain aspects, the invention provides a method of treating anautoimmune disease or a T cell leukemia in an animal in need oftreatment, comprising administering to said animal a host cellcomprising a chimeric antigen receptor targeting TCRα polypeptidecomprising sequence GSTLRG (SEQ ID NO:1).

In further embodiments, the invention provides a host cell comprisingone or more polynucleotide molecules encoding an antibody or antigenbinding fragment thereof that binds to a TCRα chain comprising GSTLRG(SEQ ID NO:1), wherein the antibody or antigen binding fragment thereofselectively binds to T-cells having a plurality of T-cell epitopespecificities.

A further embodiment provides a kit comprising an antibody of thepresent embodiments or a host cell comprising one or more polynucleotidemolecule(s) encoding an antibody that binds to a T-Cell Receptor (TCR)wherein the antibody binds to T-cells having a plurality of T-cellepitope specificities and at least a first agent for increasingproliferation of mammalian T-cells. In certain aspects, kit additionallycomprises an APC.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1A-1E(i), 1E(ii). A) Binding specificity of monoclonal antibody(Clone 79A). ELISA performed on solid phase coated with linearTCR-invariant chain specific synthetic peptides. Antibody dose curvewere run with a concentration range from 1000 ng/mL to 7.5 ng/mL.Controls include non-specific peptides, secondary antibody control andbuffer control in the assay. Assay plate was read at OD450 by Victor™plate reader. Samples were run in duplicate. B) Ala-Scan libraryconfirmed linear epitope specificity of mAb 79A on lyophilized peptides(Mimotope™ Ala-scan peptide library) coated onto solid phase ELISA.Graph shows highest binding intensity of 79A mAb upon substitution withselected AA with Alanine. C) Moderate binding of 79A to specificpeptides as shown in figure substituted with alanine. D) GSTLGR (SEQ IDNO:1) represents junction of TCR Va24 and Ja18 where 79A binding focusesas substitution of these AA by alanine completely abrogates binding.E(i) and E(ii)) 79A lacks conformational fit to TCR-CDR3 invariantchain. Flow cytometry assay shows that 79A fails to bind either purifiediNKT cell (in image) or regular αβ⁺-T cells (shown later) where asiNKT-specific mAb 6B11 detects conformational epitopes on iNKT cells.

FIG. 2. V-gene amplification of mAb clone 79A. Agarose gel image showingamplification of VH and VL chain of hybridoma clone A79. VH chain(400˜bp) shown in image after amplification using a mixture of mouseheavy chain constant region primes (MH-IgG₁) along with mouse heavychain FR1 region primers (contains a mixture of high and lowdegeneracy), and VL chain (370˜bp) after amplification using mixture ofmouse kappa chain universal degeneracy primers and kappa-chain constantprimers. All 5′ primer designed both for VL and VH starting at firstnucleotide of FR1.

FIGS. 3A-3D. A-B) Antibody (79A) homology model. Construction of 79Ahomology model based on ABR analyzed by Paratome server (Ref. Kunik, V.,Peters, B. and Ofran, Y. (2012) Structural consensus among antibodiesdefines the antigen binding site. PLoS computational biology, 8,e1002388.) (Kunik et al., 2012. Nucleic Acid Research. 40, W521-524).WAM server (on the World Wide Web at antibody.bath.ac.uk) was used tobuild VL chain and SWISS-MODEL (on the World Wide Web atswissmodel.expasy.org) was used to build VH domain because of shortVH-Chain CDR. Parameters are as per server specification. C) Z-Dockingmodel of 79A with TCR invariant chain specific peptide. Docked modelshows 79A do not induce conformation specific fit with TCR-invariantchain specific peptide derived from 2CDE. D) Docking model showing 79Aantibody in complex with αβ-TCR (PDB code 1KGC) built on web serverZDOCK and output was obtained through BuildModel function. Docked modelwas analyzed by FoldX. Ala-Scan was performed to delineate energycontribution of individual amino acids in the antibody CDR. Total changein interaction energy (ΔΔG) was calculated by subtracting interactionenergy of the wildtype from interaction energy of the mutants.

FIGS. 4A-4C. Change in interaction energy (ΔΔG) based on FoldX's complexAla-scan function. (*ΔΔG=ΔGMU−ΔGWT (−9.98)) Total interaction energy(ΔΔG) obtained from antibody docked model after Ala-scanning. ΔΔG* wascalculated by subtracting interaction energy of wild type from that ofmutants. Red circle indicates lowest change in interaction energy andresidues suitable for mutation. A) Tyrosine (Y) at position 41 of CDR1of L-chain mutated to Phenylalanine (F). Phenylalanine (F) at position94 of CDR3 of L-chain changed to Methionine (M). B) Asparitic acid (D)at position 99 of CDR3 of H-chain mutated to Alanine (A). Tryptophan (W)at position 101 of CDR3 of H-chain mutated to Leucine (L). C) Scatterline graph represents distribution of molecular interaction energy (ΔΔG)in Kcal/mole along the antibody 79A CDRs (VH and VL regions). Aminoacids with low interaction energy (<0.5 Kcal/mol) are considered ascandidate for mutation and marked (red arrow). The relevant mutationsare Serine to Glycine at position 32 (S32G), Asparagine to Glycine atposition 33 (N33G) in 79A light chain CDR1, Aspartic Acid to Glycine atposition 31 (D31G) in heavy chain CDR1, Aspartic Acid to Glycine atposition 50 (D50G) in heavy chain CDR2 and Aspartic Acid to Arginine atposition 99 (D99R) in CDR3 of 79A heavy chain. Relevant mutations wereintroduced in the 79A antibody variable regions to construct the mutantrecombinant antibody Clone S23.

FIG. 5A(i), 5A(ii), 5B. A-(i) Molecular construct for mutant recombinantantibody expression vector S15 (Single Chain Affinity optimized Novelmutant 15). Schematic shows 79A mutant scFv fused to truncated IgG₁ Fcto generate recombinant antibody. Antibody gene expression cassetteconsists of VH5 signal peptide joined in frame with variable heavy chain(VH-mutant 15) and variable light chain (VL-mutant 15). Expression isunder the control of SV40 promoter and stopped with polyA signal.Expression is under the control of SV40 promoter and ended with polyAsignal. A(ii) Vector map showing 79A mutant scFv fusions to truncatedIgG1 Fc to generate recombinant antibody (S15). Antibody expressioncassette consists of VH5 signal peptide joined in frame with variableheavy chain (VH-mutant 15) and variable light chain (VL-mutant 15).Expression is under the control of SV40 promoter and ends with polyAsignal. B) Molecular construct representing V_(H) and V_(L) of mutant 23(S23). VH variable region with modifications (D31G, D5OG and D99R) isfused to human IgG₁ Fc gamma constant chain and VL with modifications(S32G and N33G) is fused to human κ-constant light chain. Full lengthantibody sequences were PCR amplified and then cloned into MCS of a highexpression vector (Lake Pharma) before transfecting HEK293 cells fortransient expression. Culture supernatant was used for antibodypurification.

FIGS. 6A-6E. A) Homogeneity and purity of recombinant antibodies (S11,S13 and S15). Coomassie blue stained SDS-PAGE gel shows homogeneity andpurity of the recombinant antibody preparation after Protein Apurification. Shown in image are 3 different mutant antibodies expressedin mammalian 293T cells. B) 79A mutant (single chain mAb) detectsTCR-alpha chain in denatured whole T cell lysates. Whole cell lysatesextracted from donor PBMC or purified T cells run in denatured PAGE(SDS+2ME combined with 2× bromophenol blue). Equal amount (10 ug) ofprotein were loaded in each lane after normalization. Parental mAb 79Aand mutant mAbs (mutant 13 and mutant 15) were used as primary antibodyfollowed by HRP conjugated matched secondary antibody for detection.Blots were visualized by chemiluminescence and images were acquired by aBiorad imager. Maximum detection sensitivity was achieved for mutant m15with highest ΔΔG correlating “change in interaction energy” betweenparental A79 scFv and mutant mAb (S13 and S15). C) Conformation specificbinding of recombinant mAb S15 to αβ-TCR of T cells. Flow plots show S15could bind αβ-TCR⁺ T-cells in its' native configuration. Bindingimprovement in s15 (mutant 15) is correlated to the site-specificmutation introduced in 79A chains. On left panel parental mAb 79A failto bind PBMC derived T cells. However, S15 co-stains with αβ-TCR+T cellsand binding is comparable to commercial anti-αβ-TCR (WT31) antibody. D)SDS-PAGE analysis of recombinant protein (S15 and S23) run on TGXpre-stained gel (Miniprotean, Biorad). Samples were reduced with 2βMEand heat (95C) for 2 min before loading the gel. Image was acquired byBiorad imaging system (ChemiDoc). Position of both heavy and light chainof the protein A affinity purified samples were shown in picture. E) Dotplot shows improved binding of mutant recombinant antibody S15 toactivated ββ3+ T cells harvested from culture (n=3). Cells were stainedwith primary antibody 79A or S15 along with a matched fluorescentconjugated secondary antibody and run on flow cytometer as described inmethods. Live cells were gated on lymphocytes based on predefinedforward and side scatters settings on BD FACS caliber and data wereacquired for 0.5 million cells.

FIGS. 7A-7M. A) Molecular construct to tether S15 (mutantVH-linker-mutant VL) on K562-Activating and propagating cell (AaPC).Molecular construct for transfer plasmid encoding S15 (mutantVH-linker-mutant VL). Shown in image 5′ LTR (long terminal repeat) fusedto heterologous enhancer RSV Rous sarcoma virus, HIV packaging signal ψ,RRE Reverses response element, cPPT central polypurine tract, Igk leaderchain sequence, S15-VH mutant antibody variable heavy chain, S15-VLvariable light chain, 4 Glysine-3 Serine linker repeat, CD8 TMTransmembrane with HA tag and truncated CD8 Transmembrane, HBV PREWoodchuck Hepatitis virus post-transcriptional regulatory element andself-inactivating HIV LTR, Amp R ampicillin resistance gene as selectionmarker. Packaging vector (psPAX2) and envelope (pVSV-G) plasmids areadded separately. All plasmids are transfected in 293T cells to producelentiviral particles. B) Engineered K562-AaPC (K562-S15 and K562-OKT3)expresses T-cell activating and co-stimulatory ligands. Histogramshowing immunophenotype of K562 cells lentivirus transduced to expressS15 scFv (detected by anti-HA tag), T-cell co-stimulatory ligands CD86,CD137L and cytokine ligand IL15-IL15Ra. K562 cells were also engineeredto express CD64 to load CD3ε mAb OKT3, and similar co-stimulatoryligands. C) CFSE based dye dilution assay to show T cell division afteractivation via plate bound recombinant antibody S15 and S23 as comparedto CD28 and OKT3 (CD3ε specific mAb). Cells were stained for live-deadand analyzed by flow cytometry on CD3⁺ gated T cells. CFSE intensity wascaptured at 488 nm excitation by flow cytometry. Each peak representdifferent population of CD3 positive T cells generated after antibodystimulation on Day 2 D) CFSE based dye dilution assay showing T cellproliferation after plate bound antibody activation on day 6. CFSEloaded unstimulated PBMC (dark gray histogram) is used as control in theassay E) Growth kinetics of T cells expanded on K562-AaPC. Expansion ofCD3⁺ αβ-TCR⁺ T cells from healthy donor PBMC after two week co-cultureon irradiated K562-AaPC. Live cells were counted by trypan blue dyeexclusion method and represented as absolute cell number. Graph shows Tcells stimulated via S15 and OKT3 in presence of T cell co-stimulatoryligands F) Similar growth kinetics of T cells on K562 cells loaded withantibody (S15 only. K562 parental cells serve as negative control andOKT3 loaded K562 serve as positive control G) Immunophenotype of ex vivocultured activated T cells. Live activated T cells were analyzed by flowcytometry for cell surface expression of αβ-TCR (WT31), CD3, CD4 and CD8T cell markers (BD). Shown in picture pseudocolor plots describingimmunophenotype expression of markers in activated T cells harvested atday 7 of co-culture using K562 (parental), K562 S15 with co-stimulation,K562 OKT3 with co-stimulation. H) Ex vivo propagated T cells activatedby recombinant antibodies were analyzed for ab-TCR expression and ab-TCRexpression shown along with CD3. I) T cell phenotype for CD4 expressionbefore and after activation by antibodies J) T cell phenotype for CD8expression before and after activation by antibodies K) & L) T celldifferentiation markers based on expression of CD45RA and CD45RO toassess level of activation leading to generation of memory or effectormemory phenotype. Ex vivo propagated live T cells (fixable viability dyenegative) and healthy donor PBMC derived T cells were analyzed by flowcytometry. Cells were stained with CD19 and CD14 to exclude B cells andmonocytes. All live cells were gated on CD3+ CD56-negative T cellpopulation. As compared to PBMC derived unmanipulated T-cell population,there appears to be a decrease in CD4+ T cell percentage after OKT3stimulation while S23 maintains high CD4 composition, in contrast CD8percentage was highest in OKT3 stimulated group. For celldifferentiation, CD45RA negative population was highest in OKT3 groupwhile CD45RA-negative CD45RO positive population remain stable withineach group of cells irrespective of antibody clones used forstimulation. (M) Marker for T cell exhaustion PD1 is upregulated in OKT3stimulated group as compared to S15 or S23 mediated activation.

FIGS. 8A-8D. A) TCRVβ repertoire of healthy donor PBMC derived T cellscompared with activated T cells expanded on aAPC by S15 or OKT3-CD3εstimulation shows generation of population of T cells with heterogeneousT-cell repertoire. No unexpected skewing of repertoire in activated invitro expanded T cells are noticed. B) TCRVβ repertoire of activated Tcells grown by either S15 stimulation (CS1S15) of by OKT3 T cells. Therepertoire represents a polyclonal T cell population without anyspecific growth of clonotypes. C) Next generation deep sequence surveyof T cell clone frequency and distribution after ex vivo propagation.Commonality in clonotypes are seen to be aligned with PBMC better afterS15 mediated activation as compared to OKT3 stimulation D) Table showsPearson coefficient values (r²) representing clonal frequency of T cellsgenerated after activation. T cell clone frequencies are comparedbetween T cells stimulated with (Parental K562/ACT), with(K562-S15-Co-stims) and (K562-OKT3-Co-stims). Sample overlap as comparedto PBMC shows r² values 0.043 and o=0.472 for S15 T cells and r²=0.055and sample overlap o=0.172 for OKT3-PBMC. Clonal overlap is superior forS15 activated T cells.

FIG. 9. V-J Paired gene frequency. Stalked histograms show V-J pairedgene frequency in any particular locus for T cells grown on K562-S15with co-stimulation (clone CS1S15) and for T cells grown on K562-OKT3(Clone K562 OKT3) with co-stimulation.

FIG. 10. Clonal hierarchy of unmanipulated T cells versus activated Tcells as analyzed from NGS data after deep sequencing. Clones in highestranking (1-10) based on percentage of productive sequencing readsrepresented by each clone in healthy donor PBMC sample used for T cellactivation and propagation (top panel). Table shows amino acid sequencesof corresponding TCR CDR3, along with V and J family alleles andrespective percentage. A comparison of top clone frequency between Tcells activated by K562-S15 vs. K562-OKT3 are shown as histograms (lowerpanels). Y-axis represents percentage of total counts per samples.

FIG. 11. Bell curve distribution of CDR3 chain length both for S15activated T cells and OKT3 activated T cells. CDR3 chain length ofactivated T cells when compared to PBMC-derived T cells, no perturbationover all distribution of CDR3 chain is observed.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS I. DEFINITIONS

As used herein, “essentially free,” in terms of a specified component,is used herein to mean that none of the specified component has beenpurposefully formulated into a composition and/or is present only as acontaminant or in trace amounts. The total amount of the specifiedcomponent resulting from any unintended contamination of a compositionis therefore preferably below 0.01%. Most preferred is a composition inwhich no amount of the specified component can be detected with standardanalytical methods.

As used herein in the specification and claims, “a” or “an” may mean oneor more. As used herein in the specification and claims, when used inconjunction with the word “comprising”, the words “a” or “an” may meanone or more than one. As used herein, in the specification and claim,“another” or “a further” may mean at least a second or more.

As used herein in the specification and claims, the term “about” is usedto indicate that a value includes the inherent variation of error forthe device, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids, are includedwithin the definition of “polypeptide,” and the term “polypeptide” maybe used instead of or interchangeably with any of these terms. The term“polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide may be derived from a natural biological source or producedby recombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It may be generated in any manner,including by chemical synthesis.

A polypeptide of the invention may be of a size of about 3 or more, 5 ormore, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 ormore, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides may have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure are referred to as folded, andpolypeptides that do not possess a defined three-dimensional structure,but rather can adopt a large number of different conformations, arereferred to as unfolded. As used herein, the term glycoprotein refers toa protein coupled to at least one carbohydrate moiety that is attachedto the protein via an oxygen-containing or a nitrogen-containing sidechain of an amino acid residue, e.g., a serine residue or an asparagineresidue.

By an “isolated” polypeptide or a fragment, variant, or derivativethereof is intended a polypeptide that is not in its natural milieu. Noparticular level of purification is required. For example, an isolatedpolypeptide can be removed from its native or natural environment.Synthetic recombinant polypeptides and/or proteins expressed in hostcells are considered isolated for purpose of the invention, as arenative or recombinant polypeptides that have been separated,fractionated, or partially or substantially purified by any suitabletechnique.

Also included as polypeptides of the present invention are fragments,derivatives, analogs, or variants of the foregoing polypeptides, and anycombination thereof. The terms “fragment,” “variant,” “derivative,” and“analog” when referring to anti-TCR antibodies or antibody polypeptidesof the present invention include any polypeptides that retain at leastsome of the antigen-binding properties of the corresponding antibody orantibody polypeptide of the invention. Fragments of polypeptides of thepresent invention include proteolytic fragments, as well as deletionfragments, in addition to specific antibody fragments discussedelsewhere herein. Variants of anti-TCR antibodies and antibodypolypeptides of the present invention include fragments as describedabove, and also polypeptides with altered amino acid sequences due toamino acid substitutions, deletions, or insertions. Variants may occurnaturally or be non-naturally occurring. Non-naturally occurringvariants may be produced using art-known mutagenesis techniques. Variantpolypeptides may comprise conservative or non-conservative amino acidsubstitutions, deletions, or additions. Variant polypeptides may also bereferred to herein as “polypeptide analogs,” As used herein a“derivative” of an anti-TCR antibody or antibody polypeptide refers to asubject polypeptide having one or more residues chemically derivatizedby reaction of a functional side group. Also included as “derivatives”are those peptides that contain one or more naturally occurring aminoacid derivatives of the twenty standard amino acids. For example,4-hydroxyproline may be substituted for proline; 5-hydroxylysine may besubstituted for lysine; 3-methylhistidine may be substituted forhistidine; homoserine may be substituted for serine; and ornithine maybe substituted for lysine. Derivatives of anti-TCR antibodies andantibody polypeptides of the present invention, may include polypeptidesthat have been altered so as to exhibit additional features not found onthe reference antibody or antibody polypeptide of the invention.

The term “polynucleotide” is intended to encompass a singular nucleicacid as well as plural nucleic acids, and refers to an isolated nucleicacid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA(pDNA). A polynucleotide may comprise a conventional phosphodiester bondor a non-conventional bond (e.g., an amide bond, such as found inpeptide nucleic acids (PNA)). The term “nucleic acid” refers to any oneor more nucleic acid segments, e.g., DNA or RNA fragments, present in apolynucleotide. By “isolated” nucleic acid or polynucleotide is intendeda nucleic acid molecule, DNA or RNA, that has been removed from itsnative environment. For example, a recombinant polynucleotide encodingan anti-TCR binding molecule, e.g., an antibody or antigen bindingfragment thereof, contained in a vector is considered isolated for thepurposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. Isolated RNA molecules include in vivo orin vitro RNA transcripts of polynucleotides of the present invention.Isolated polynucleotides or nucleic acids according to the presentinvention further include such molecules produced synthetically. Inaddition, a polynucleotide or a nucleic acid may be or may include aregulatory element such as a promoter, ribosome binding site, or atranscription terminator.

As used herein, a “coding region” is a portion of nucleic acid thatconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it may beconsidered to be part of a coding region, but any flanking sequences,for example promoters, ribosome binding sites, transcriptionalterminators, introns, and the like, are not part of a coding region. Twoor more coding regions of the present invention can be present in asingle polynucleotide construct, e.g., on a single vector, or inseparate polynucleotide constructs, e.g., on separate (different)vectors. Furthermore, any vector may contain a single coding region, ormay comprise two or more coding regions, e.g., a single vector mayseparately encode an immunoglobulin heavy chain variable region and animmunoglobulin light chain variable region. In addition, a vector,polynucleotide, or nucleic acid of the invention may encode heterologouscoding regions, either fused or unfused to a nucleic acid encoding ananti-TCR antibody or fragment, variant, or derivative thereof.Heterologous coding regions include without limitation specializedelements or motifs, such as a secretory signal peptide or a heterologousfunctional domain.

In certain embodiments, the polynucleotide or nucleic acid is DNA. Inthe case of DNA, a polynucleotide comprising a nucleic acid that encodesa polypeptide normally may include a promoter and/or other transcriptionor translation control elements operably associated with one or morecoding regions. An operable association is when a coding region for agene product, e.g., a polypeptide, is associated with one or moreregulatory sequences in such a way as to place expression of the geneproduct under the influence or control of the regulatory sequence(s).Two DNA fragments (such as a polypeptide coding region and a promoterassociated therewith) are “operably associated” if induction of promoterfunction results in the transcription of mRNA encoding the desired geneproduct and if the nature of the linkage between the two DNA fragmentsdoes not interfere with the ability of the expression regulatorysequences to direct the expression of the gene product or interfere withthe ability of the DNA template to be transcribed. Thus, a promoterregion would be operably associated with a nucleic acid encoding apolypeptide if the promoter was capable of effecting transcription ofthat nucleic acid. The promoter may be a cell-specific promoter thatdirects substantial transcription of the DNA only in predeterminedcells. Other transcription control elements, besides a promoter, forexample enhancers, operators, repressors, and transcription terminationsignals, can be operably associated with the polynucleotide to directcell-specific transcription. Suitable promoters and other transcriptioncontrol regions are disclosed herein.

A variety of transcription control regions are known to those skilled inthe art. These include, without limitation, transcription controlregions that function in vertebrate cells, such as, but not limited to,promoter and enhancer segments from cytomegaloviruses (the immediateearly promoter, in conjunction with intron-A), simian virus 40 (theearly promoter), and retroviruses (such as Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit .beta.-globin, as well as other sequences capable of controllinggene expression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins).

Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited to,ribosome binding sites, translation initiation and termination codons,and elements derived from picornaviruses (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence).

In other embodiments, a polynucleotide of the present invention is RNA,for example, in the form of messenger RNA (mRNA).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions that encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. According to the signalhypothesis, proteins secreted by mammalian cells have a signal peptideor secretory leader sequence that is cleaved from the mature proteinonce export of the growing protein chain across the rough endoplasmicreticulum has been initiated. Those of ordinary skill in the art areaware that polypeptides secreted by vertebrate cells generally have asignal peptide fused to the N-terminus of the polypeptide, which iscleaved from the complete or “full length” polypeptide to produce asecreted or “mature” form of the polypeptide. In certain embodiments,the native signal peptide, e.g., an immunoglobulin heavy chain or lightchain signal peptide is used, or a functional derivative of thatsequence that retains the ability to direct the secretion of thepolypeptide that is operably associated with it. Alternatively, aheterologous mammalian signal peptide, or a functional derivativethereof, may be used. For example, the wild-type leader sequence may besubstituted with the leader sequence of human tissue plasminogenactivator (TPA) or mouse β-glucoronidase.

As used herein, the term “TCR” refers to “T cell receptor.” A T cellreceptor is a molecule on the surface of T lymphocytes (“T cells”). Inembodiments, the receptor is an αβ-TCR receptor, meaning that the T cellreceptor comprises an alpha (α) and beta (β) chain, which is typicallyexpressed as part of a complex with CD3 chain molecules. Thus, an“αβ-TCR⁺ T cell” is a T lymphocyte that contains a T cell receptor onits surface that comprises α and β chains. Both the α and β chains arehighly variable, although the T-cell receptor a chain contains aconstant (preserved region), i.e., the Va24-Ja18 junction (amino acidsequence GSTLGR (SEQ ID NO:1)).

A “binding molecule” or “antigen binding molecule” of the presentinvention refers in its broadest sense to a molecule that specificallybinds an antigenic determinant. In one embodiment, the binding moleculespecifically binds to TCR, e.g., a T-cell receptor, e.g., a T-cellreceptor α chain. In embodiments, the binding molecule specificallybinds to the Va24-Ja18 junction (amino acid sequence GSTLGR (SEQ IDNO:1)) of the T-cell receptor α-chain. In another embodiment, a bindingmolecule of the invention is an antibody or an antigen binding fragmentthereof that includes point mutations to increase affinity. In anotherembodiment, a binding molecule of the invention comprises at least oneheavy or light chain CDR of an antibody molecule. In another embodiment,a binding molecule of the invention comprises at least two CDRs from oneor more antibody molecules. In another embodiment, a binding molecule ofthe invention comprises at least three CDRs from one or more antibodymolecules. In another embodiment, a binding molecule of the inventioncomprises at least four CDRs from one or more antibody molecules. Inanother embodiment, a binding molecule of the invention comprises atleast five CDRs from one or more antibody molecules. In anotherembodiment, a binding molecule of the invention comprises at least sixCDRs from one or more antibody molecules.

The present invention is directed to certain anti-TCR antibodies, orantigen-binding fragments, variants, or derivatives thereof. Unlessspecifically referring to a full length antibodies such as naturallyoccurring antibodies, the term “anti-TCR antibodies” encompassesantigen-binding fragments, variants, analogs, or derivatives of suchantibodies, e.g., naturally occurring antibody or immunoglobulinmolecules or engineered antibody molecules or fragments that bindantigen in a manner similar to antibody molecules.

As used herein, “human” or “fully human” antibodies include antibodieshaving the amino acid sequence of a human immunoglobulin and includeantibodies isolated from human immunoglobulin libraries or from animalstransgenic for one or more human immunoglobulins and that do not expressendogenous immunoglobulins, as described infra and, for example, in U.S.Pat. No. 5,939,598 by Kucherlapati et “Human” or “fully human”antibodies also include antibodies comprising at least the variabledomain of a heavy chain, or at least the variable domains of a heavychain and a light chain, where the variable domain(s) have the aminoacid sequence of human immunoglobulin variable domain(s).

“Human” or “fully human” antibodies also include “human” or “fullyhuman” antibodies, as described above, that comprise, consistessentially of, or consist of, variants (including derivatives) ofantibody molecules (e.g., the VH regions and/or VL regions) describedherein, which antibodies or fragments thereof immunospecifically bind toa TCR or fragment or variant thereof. Standard techniques known to thoseof skill in the art can be used to introduce mutations in the nucleotidesequence encoding a human anti-TCR antibody, including, but not limitedto, site-directed mutagenesis and PCR-mediated mutagenesis which resultin amino acid substitutions. Preferably, the variants (includingderivatives) encode less than 50 amino acid substitutions, less than 40amino acid substitutions, less than 30 amino acid substitutions, lessthan 25 amino acid substitutions, less than 20 amino acid substitutions,less than 15 amino acid substitutions, less than 10 amino acidsubstitutions, less than 5 amino acid substitutions, less than 4 aminoacid substitutions, less than 3 amino acid substitutions, or less than 2amino acid substitutions relative to the reference VH region, HCDR1.HCDR2, HCDR3, VL region, LCDR1, LCDR2, or LCDR3.

In certain embodiments, the amino acid substitutions are conservativeamino acid substitution, discussed further below. Alternatively,mutations can be introduced randomly along all or part of the codingsequence, such as by saturation mutagenesis, and the resultant mutantscan be screened for biological activity to identify mutants that retainactivity (e.g., the ability to bind a TCR polypeptide, e.g., human,murine, or both human and murine TCR). Such variants (or derivativesthereof) of “human” or “fully human” antibodies can also be referred toas human or fully human antibodies that are “optimized” or “optimizedfor antigen binding” and include antibodies that have improved affinityto antigen.

The terms “antibody” and “immunoglobulin” are used interchangeablyherein. An antibody or immunoglobulin comprises at least the variabledomain of a heavy chain, and normally comprises at least the variabledomains of a heavy chain and a light chain. Basic immunoglobulinstructures in vertebrate systems are relatively well understood. See,e.g., Harlow et al. (1988) Antibodies: A Laboratory Manual (2nd ed.;Cold Spring Harbor Laboratory Press).

As will be discussed in more detail below, the term “immunoglobulin”comprises various broad classes of polypeptides that can bedistinguished biochemically. Those skilled in the art will appreciatethat heavy chains are classified as gamma, mu, alpha, delta, or epsilon,with some subclasses among them (e.g., gammal-gamma4). It is the natureof this chain that determines the “class” of the antibody as IgG, IgM,IgA IgG, or IgE, respectively. The immunoglobulin subclasses (isotypes)e.g., IgG1, IgG2, IgG3, IgG4, IgAl, etc. are well characterized and areknown to confer functional specialization. Modified versions of each ofthese classes and isotypes are readily discernable to the skilledartisan in view of the instant disclosure and, accordingly, are withinthe scope of the instant invention. All immunoglobulin classes areclearly within the scope of the present invention. The followingdiscussion will generally be directed to the IgG class of immunoglobulinmolecules. With regard to IgG, a standard immunoglobulin moleculecomprises two identical light chain polypeptides of molecular weightapproximately 23,000 Daltons, and two identical heavy chain polypeptidesof molecular weight 53,000-70,000. The four chains are typically joinedby disulfide bonds in a “Y” configuration wherein the light chainsbracket the heavy chains starting at the mouth of the “Y” and continuingthrough the variable region.

Light chains are classified as either kappa or lambda (κ, λ). Each heavychain class may be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (VL or VK) and heavy (VH) chain portionsdetermine antigen recognition and specificity. Conversely, the constantdomains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3)confer important biological properties such as secretion, transplacentalmobility, Fc receptor binding, complement binding, and the like. Byconvention the numbering of the constant region domains increases asthey become more distal from the antigen binding site or amino-terminusof the antibody. The N-terminal portion is a variable region and at theC-terminal portion is a constant region; the CH3 and CL domains actuallycomprise the carboxy-terminus of the heavy and light chain,respectively.

As indicated above, the variable region allows the antibody toselectively recognize and specifically bind epitopes on antigens. Thatis, the VL domain and VH domain, or subset of the complementaritydetermining regions (CDRs) within these variable domains, of an antibodycombine to form the variable region that defines a three-dimensionalantigen binding site. This quaternary antibody structure forms theantigen binding site present at the end of each arm of the Y. Morespecifically, the antigen binding site is defined by three CDRs on eachof the VH and VL chains. As used herein, the terms HCDR1, HCDR2, HCDR3refer to VH CDR1, VH CDR2, VH CDR3, respectively. Likewise, as usedherein, the terms LCDR1, LCDR2, LCDR3, refer to VL CDR1, VL CDR2, and VLCDR3, respectively. In some instances, e.g., certain immunoglobulinmolecules derived from camelid species or engineered based on camelidimmunoglobulins, a complete immunoglobulin molecule may consist of heavychains only, with no light chains. See, e.g., Hamers-Casterman et al.,Nature 363:446-448 (1993).

In naturally occurring antibodies, the six “complementarity determiningregions” or “CDRs” present in each antigen binding domain are short,non-contiguous sequences of amino acids that are specifically positionedto form the antigen binding domain as the antibody assumes itsthree-dimensional configuration in an aqueous environment. The remainderof the amino acids in the antigen binding domains, referred to as“framework” regions, show less inter-molecular variability. Theframework regions largely adopt a sheet conformation and the CDRs formloops that connect, and in some cases form part of, the β-sheetstructure. Thus, framework regions act to form a scaffold that providesfor positioning the CDRs in correct orientation by inter-chain,non-covalent interactions. The antigen binding domain formed by thepositioned CDRs defines a surface complementary to the epitope on theimmunoreactive antigen. This complementary surface promotes thenon-covalent binding of the antibody to its cognate epitope. The aminoacids comprising the CDRs and the framework regions, respectively, canbe readily identified for any given heavy or light chain variable domainby one of ordinary skill in the art, since they have been preciselydefined (see below).

In the case where there are two or more definitions of a term that isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. This particular region hasbeen described by Kabat et al. (1983) U.S. Dept. of Health and HumanServices, “Sequences of Proteins of Immunological Interest,” by Chothiaand Lesk, J. Mol. Biol. 196:901-917 (1987), and updated recently byKunik et al., Nucl. Acids Res. 40:W521-W524 (2012), which areincorporated herein by reference, where the definitions includeoverlapping or subsets of amino acid residues when compared against eachother. Nevertheless, application of any definition to refer to a CDR ofan antibody or variants thereof is intended to be within the scope ofthe term as defined and used herein. The appropriate amino acid residuesthat encompass the CDRs as defined by each of the above cited referencesare set forth below in Table 1 as a comparison. The exact residuenumbers that encompass a particular CDR will vary depending on thesequence and size of the CDR. Those skilled in the art can routinelydetermine which residues comprise a particular CDR given the variableregion amino acid sequence of the antibody.

Kabat et al. also defined a numbering system for variable domainsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable domain sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al. (1983) U.S. Dept. ofHealth and Human Services, “Sequence of Proteins of ImmunologicalInterest.”

Kunik et al., Nucl. Acids Res. 40:W521-W524 (2012) disclosed an onlinetool, Paratome, for systematic identification of antigen-binding regionsin antibodies based on sequence or structure. Usually the Paratome-basedanalysis matches with Kabat numbering, but may also include residuesadjacent to conventional CDRs. Unless otherwise specified, references tothe numbering of specific amino acid residue positions in an anti-TCRantibody CDRs or antigen-binding fragment, variant, or derivativethereof of the present invention are according to the numbering systembased on Paratome identification.

Antibodies or antigen-binding fragments, variants, or derivativesthereof of the invention include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized, primatized, or chimericantibodies, single-chain antibodies, epitope-binding fragments, e.g.,Fab, Fab′ and F(ab′)₂, Fd, Fvs, single-chain Fvs (scFv),disulfide-linked Fvs (sdFv), fragments comprising either a VL or VHdomain, fragments produced by a Fab expression library, andanti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodiesto anti-TCR antibodies disclosed herein). ScFv molecules are known inthe art and are described, e.g., in U.S. Pat. No. 5,892,019.Immunoglobulin or antibody molecules of the invention can be of any typeIgG, IgE, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1,and IgA2, etc.), or subclass of immunoglobulin molecule.

As used herein, the term “heavy chain portion” includes amino acidsequences derived from an immunoglobulin heavy chain. A polypeptidecomprising a heavy chain portion comprises at least one of: a CH1domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain,a CH2 domain, a CH3 domain, or a variant or fragment thereof. Forexample, a binding polypeptide for use in the invention may comprise apolypeptide chain comprising a CH1 domain; a polypeptide chaincomprising a CH1 domain, at least a portion of a hinge domain, and a CH2domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; apolypeptide chain comprising a CH1 domain, at least a portion of a hingedomain, and a CH3 domain, or a polypeptide chain comprising a CH1domain, at least a portion of a hinge domain, a CH2 domain, and a CH3domain. In another embodiment, a polypeptide of the invention comprisesa polypeptide chain comprising a CH3 domain. Further, a bindingpolypeptide for use in the invention may lack at least a portion of aCH2 domain (e.g., all or part of a CH2 domain). As set forth above, itwill be understood by one of ordinary skill in the art that thesedomains (e.g., the heavy chain portions) may be modified such that theyvary in amino acid sequence from the naturally occurring immunoglobulinmolecule.

In certain anti-TCR antibodies, or antigen-binding fragments, variants,or derivatives thereof disclosed herein, the heavy chain portions of onepolypeptide chain of a multimer are identical to those on a secondpolypeptide chain of the multimer. Alternatively, heavy chainportion-containing monomers of the invention are not identical. Forexample, each monomer may comprise a different target binding site,forming, for example, a bispecific antibody.

The heavy chain portions of a binding molecule for use in the diagnosticand treatment methods disclosed herein may be derived from differentimmunoglobulin molecules. For example, a heavy chain portion of apolypeptide may comprise a C.sub.H1 domain derived from an IgG1 moleculeand a hinge region derived from an IgG3 molecule. In another example, aheavy chain portion can comprise a hinge region derived, in part, froman IgG1 molecule and, in part, from an IgG3 molecule. In anotherexample, a heavy chain portion can comprise a chimeric hinge derived, inpart, from an IgG1 molecule and, in part, from an IgG4 molecule.

As used herein, the term “light chain portion” includes amino acidsequences derived from an immunoglobulin light chain, e.g., a kappa orlambda light chain. Preferably, the light chain portion comprises atleast one of a VL or CL domain.

Anti-TCR antibodies, or antigen-binding fragments, variants, orderivatives thereof disclosed herein may be described or specified interms of the epitope(s) or portion(s) of an antigen, e.g., a targetpolypeptide disclosed herein (e.g., TCR) that they recognize orspecifically bind. The portion of a target polypeptide that specificallyinteracts with the antigen binding domain of an antibody is an“epitope,” or an “antigenic determinant.” A target polypeptide maycomprise a single epitope, but typically comprises at least twoepitopes, and can include any number of epitopes, depending on the size,conformation, and type of antigen. Furthermore, it should be noted thatan “epitope” on a target polypeptide may be or may includenon-polypeptide elements, e.g., an epitope may include a carbohydrateside chain.

The minimum size of a peptide or polypeptide epitope for an antibody, isthought to be about four to five amino acids. Peptide or polypeptideepitopes preferably contain at least seven, more preferably at leastnine and most preferably between at least about 15 to about 30 aminoacids. Since a CDR can recognize an antigenic peptide or polypeptide inits tertiary form, the amino acids comprising an epitope need not becontiguous, and in some cases, may not even be on the same peptidechain. A peptide or polypeptide epitope recognized by anti-TCRantibodies of the present invention may contain a sequence of at least4, at least 5, at least 6, at least 7, more preferably at least 8, atleast 9, at least 10, at least 15, at least 20, at least 25, or betweenabout 15 to about 30 contiguous or non-contiguous amino acids of TCR.

By “specifically binds,” it is generally meant that an antibody binds toan epitope via its antigen binding domain, and that the binding entailssome complementarity between the antigen binding domain and the epitope.According to this definition, an antibody is said to “specifically bind”to an epitope when it binds to that epitope, via its antigen bindingdomain more readily than it would bind to a random, unrelated epitope.The term “specificity” is used herein to quality the relative affinityby which a certain antibody binds to a certain epitope. For example,antibody “A” may be deemed to have a higher specificity for a givenepitope than antibody “B,” or antibody “A” may be said to bind toepitope “C” with a higher specificity than it has for related epitope“D.”

By “preferentially binds,” it is meant that the antibody specificallybinds to an epitope more readily than it would bind to a related,similar, homologous, or analogous epitope. Thus, an antibody that“preferentially binds” to a given epitope would more likely bind to thatepitope than to a related epitope, even though such an antibody maycross-react with the related epitope.

By way of non-limiting example, an antibody may be considered to bind afirst epitope preferentially if it binds said first epitope with adissociation constant (K_(D)) that is less than the antibody's K_(D) forthe second epitope. In another non-limiting example, an antibody may beconsidered to bind a first antigen preferentially if it binds the firstepitope with an affinity that is at least one order of magnitude lessthan the antibody's K_(D) for the second epitope. In anothernon-limiting example, an antibody may be considered to bind a firstepitope preferentially if it binds the first epitope with an affinitythat is at least two orders of magnitude less than the antibody's K_(D)for the second epitope.

In another non-limiting example, an antibody may be considered to bind afirst epitope preferentially if it binds the first epitope with an offrate (k(off)) that is less than the antibody's k(off) for the secondepitope. In another non-limiting example, an antibody may be consideredto bind a first epitope preferentially if it binds the first epitopewith an affinity that is at least one order of magnitude less than theantibody's k(off) for the second epitope. In another non-limitingexample, an antibody may be considered to bind a first epitopepreferentially if it binds the first epitope with an affinity that is atleast two orders of magnitude less than the antibody's k(off) for thesecond epitope. An antibody or antigen-binding fragment, variant, orderivative disclosed herein may be said to bind a target polypeptidedisclosed herein (e.g., TCR, e.g., human, murine, or both human andmurine TCR) or a fragment or variant thereof with an off rate (k(off))of less than or equal to 5×10⁻² sec⁻¹, 10⁻³ sec⁻¹, 5×10⁻³ sec⁻¹ or 10⁻³sec⁻¹. More preferably, an antibody of the invention may be said to binda target polypeptide disclosed herein (e.g., TCR, e.g., human, murine,or both human and murine TCR) or a fragment or variant thereof with anoff rate (k(off)) less than or equal to 5×10⁻⁴ sec⁻¹, 10⁻⁴ sec⁻¹, 5×10⁻⁵sec⁻¹, or 10⁻⁵ sec⁻¹, 5×10⁻⁶ sec⁻¹, 10⁻⁶ sec⁻¹, 5×10⁻⁷ sec⁻¹ or 10⁻⁷sec⁻¹.

Anti-TCR antibodies or antigen binding fragments, variants orderivatives thereof of the invention may be “multispecific,” bispecific,trispecific, or of greater muitispecificity, meaning that it recognizesand binds to two or more different epitopes present on one or moredifferent antigens (e.g., proteins) at the same time. Thus, whether ananti-TCR antibody is “monospecific” or “multispecific,” e.g.,“bispecific,” refers to the number of different epitopes with which abinding polypeptide reacts. Multispecific antibodies may be specific fordifferent epitopes of a target polypeptide described herein or may bespecific for a target polypeptide as well as for a heterologous epitope,such as a heterologous polypeptide or solid support material.

As used herein the term “valency” refers to the number of potentialbinding domains, e.g., antigen binding domains present in a bindingpolypeptide or TCR binding molecule, e.g., an antibody or antigenbinding fragment thereof. Each binding domain specifically binds oneepitope. When a binding polypeptide or TCR binding molecule comprisesmore than one binding domain, each binding domain may specifically bindthe same epitope, for an antibody with two binding domains, termed“bivalent monospecific,” or to different epitopes, for an antibody withtwo binding domains, termed “bivalent bispecific.” An antibody orantigen binding fragment thereof may also be bispecific and bivalent foreach specificity (termed “bispecific tetravalent antibodies”). Inanother embodiment, tetravalent minibodies or domain deleted antibodiescan be made.

Bispecific bivalent antibodies, and methods of making them, aredescribed, for instance in U.S. Pat. Nos. 5,731,168; 5,807,706;5,821,333; and U.S. Patent Appl. Publ. Nos. 2003/020734 and2002/0155537, the disclosures of all of which are incorporated byreference herein. Bispecific tetravalent antibodies, and methods ofmaking them are described, for instance, in WO 02/096948 and WO00/14788, the disclosures of both of which are incorporated by referenceherein. See generally, PCT publications WO 93/17715; WO 92/08802; WO91/00360; WO 92/05793; Tutt et al., J. Immunol. 147:60-69 (1991); U.S.Pat. Nos. 4,471,893; 4,714,681; 4,925,618; 5,573,920; 5,601,819;Kostelny et al., J. Immunol. 148: 1547-1553 (1992).

As previously indicated, the subunit structures and three-dimensionalconfiguration of the constant regions of the various immunoglobulinclasses are well known. As used herein, the term “VH domain” includesthe amino terminal variable domain of an immunoglobulin heavy chain andthe term “CH1 domain” includes the first (most amino terminal) constantregion domain of an immunoglobulin heavy chain. The CH1 domain isadjacent to the VH domain and is amino terminal to the hinge region ofan immunoglobulin heavy chain molecule.

As used herein the term “CH2 domain” includes the portion of a heavychain molecule that extends, e.g., from about residue 244 to residue 360of an antibody using conventional numbering schemes (residues 244 to360, Kabat numbering system; and residues 231-340, EU numbering system;see Kabat E A et al.). The CH2 domain is unique in that it is notclosely paired with another domain. Rather, two N-linked branchedcarbohydrate chains are interposed between the two CH2 domains of anintact native IgG molecule. It is also well documented that the CH3domain extends from the CH2 domain to the C-terminal of the IgG moleculeand comprises approximately 108 residues.

As used herein, the term “hinge region” includes the portion of a heavychain molecule that joins the CH1 domain to the CH2 domain. This hingeregion comprises approximately 25 residues and is flexible, thusallowing the two N-terminal antigen binding regions to moveindependently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains (Roux et al., J.Immunol. 161:4083 (1998)).

As used herein the term “disulfide bond” includes the covalent bondformed between two sulfur atoms. The amino acid cysteine comprises athiol group that can form a disulfide bond or bridge with a second thiolgroup. In most naturally occurring IgG molecules, the CH1 and CL regionsare linked by a disulfide bond and the two heavy chains are linked bytwo disulfide bonds at positions corresponding to 239 and 242 using thekabat numbering system (position 226 or 229, EU numbering system).

As used herein, the term “chimeric antibody” will be held to mean anyantibody wherein the immunoreactive region or site is obtained orderived from a first species and the constant region (which may beintact, partial or modified in accordance with the instant invention) isobtained from a second species. In embodiments, the target bindingregion or site will be from anon-human source mouse or primate) and theconstant region is human.

As used herein, the term “engineered antibody” refers to an antibody inwhich the variable domain in either the heavy or light chain or both isaltered by at least partial replacement of one or more CDRs from, anantibody of known specificity and, if necessary, by partial frameworkregion replacement and sequence changing. Although the CDRs may bederived from an antibody of the same class or even subclass as theantibody from which the framework regions are derived, it is envisagedthat the CDRs will be derived from an antibody of different class andpreferably from an antibody from a different species. An engineeredantibody in which one or more “donor” CDRs from a non-human antibody ofknown specificity is grafted into a human heavy or light chain frameworkregion is referred to herein as a “humanized antibody.” It may not benecessary to replace all of the CDRs with the complete CDRs from thedonor variable domain to transfer the antigen binding capacity of onevariable domain to another. Rather, it may only be necessary to transferthose residues that are necessary to maintain the activity of the targetbinding site.

It is further recognized that the framework regions within the variabledomain in a heavy or light chain, or both, of a humanized antibody maycomprise solely residues of human origin, in which case these frameworkregions of the humanized antibody are referred to as “fully humanframework regions.” Alternatively, one or more residues of the frameworkregion(s) of the donor variable domain can be engineered within thecorresponding position of the human framework region(s) of a variabledomain in a heavy or light chain, or both, of a humanized antibody ifnecessary to maintain proper binding or to enhance binding to the TCRantigen. A human framework region that has been engineered in thismanner would thus comprise a mixture of human and donor frameworkresidues, and is referred to herein as a “partially human frameworkregion.”

For example, humanization of an anti-TCR antibody can be essentiallyperformed following the method of Winter and co-workers (Jones et al.,Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988);Verhoeyen et al., Science 239:1534-1536 (1988)), by substituting rodentor mutant rodent CDRs or CDR sequences for the corresponding sequencesof a human anti-TCR antibody. See also U.S. Pat. Nos. 5,225,539;5,585,089; 5,693,761; 5,693,762; 5,859,205; herein incorporated byreference. The resulting humanized anti-TCR antibody would comprise atleast one rodent or mutant rodent CDR within the fully human frameworkregions of the variable domain of the heavy and/or light chain of thehumanized antibody. In some instances, residues within the frameworkregions of one or more variable domains of the humanized anti-TCRantibody are replaced by corresponding non-human (for example, rodent)residues (see, for example, U.S. Pat. Nos. 5,585,089; 5,693,761;5,693,762; and 6,180,370), in which case the resulting humanizedanti-TCR antibody would comprise partially human framework regionswithin the variable domain of the heavy and/or light chain.

Furthermore, humanized antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance (e.g., toobtain desired affinity). In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDRs correspond tothose of a non-human immunoglobulin and all or substantially all of theframework regions are those of a human immunoglobulin sequence. Thehumanized antibody optionally also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature331:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Strict. Biol. 2:593-596 (1992); herein incorporated byreference. Accordingly, such “humanized” antibodies may includeantibodies wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some frameworkresidues are substituted by residues from analogous sites in rodentantibodies. See, for example, U.S. Pat. Nos. 5,225,539; 5,585,089;5,693,761; 5,693,762; 5,859,205. See also U.S. Pat. No. 6,180,370, andInternational Publication No. WO 01/27160, where humanized antibodiesand techniques for producing humanized antibodies having improvedaffinity for a predetermined antigen are disclosed.

As used herein, the terms “linked,” “fused,” or “fusion” are usedinterchangeably. These terms refer to the joining together of two moreelements or components, by whatever means including chemical conjugationor recombinant means. An “in-frame fusion” refers to the joining of twoor more polynucleotide open reading frames (ORFs) to form a continuouslonger ORF, in a manner that maintains the correct translational readingframe of the original ORFs. Thus, a recombinant fusion protein is asingle protein containing two or more segments that correspond topolypeptides encoded by the original ORFs (which segments are notnormally so joined in nature). Although the reading frame is thus madecontinuous throughout the fused segments, the segments may be physicallyor spatially separated by, for example, in-frame linker sequence. Forexample, polynucleotides encoding the CDRs of an immunoglobulin variableregion may be fused, in-frame, but be separated by a polynucleotideencoding at least one immunoglobulin framework region or additional CDRregions, as long as the “fused” CDRs are co-translated as part of acontinuous polypeptide.

In the context of polypeptides, a “linear sequence” or a “sequence” isan order of amino acids in a polypeptide in an amino to carboxylterminal direction in which residues that neighbor each other in thesequence are contiguous in the primary structure of the polypeptide.

The term “expression” as used herein refers to a process by which a geneproduces a biochemical, for example, a polypeptide. The process includesany manifestation of the functional presence of the gene within the cellincluding, without limitation, gene knockdown as well as both transientexpression and stable expression. It includes without limitationtranscription of the gene into messenger RNA (mRNA), and the translationof such mRNA into polypeptide(s). If the final desired product is abiochemical, expression includes the creation of that biochemical andany precursors. Expression of a gene produces a “gene product.” As usedherein, a gene product can be either a nucleic acid, e.g., a messengerRNA produced by transcription of a gene, or a polypeptide which istranslated from a transcript. Gene products described herein furtherinclude nucleic acids with post transcriptional modifications, e.g.,polyadenylation, or polypeptides with post translational modifications,e.g., methylation, glycosylation, the addition of lipids, associationwith other protein subunits, proteolytic cleavage, and the like.

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the progression of multiplesclerosis, arthritis, or cancer. Beneficial or desired clinical resultsinclude, but are not limited to, alleviation of symptoms, diminishmentof extent of disease, stabilized (i.e., not worsening) state of disease,delay or slowing of disease progression, amelioration or palliation ofthe disease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Those in need of treatment include those already with the condition ordisorder as well as those prone to have the condition or disorder orthose in which the condition or disorder is to be prevented.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. Mammalian subjects include humans,domestic animals, farm animals, and zoo, sports, or pet animals such asdogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, andso on.

As used herein, phrases such as “a subject that would benefit fromadministration of an anti-TCR antibody” and “an animal in need oftreatment” and “a subject in need thereof” includes subjects, such asmammalian subjects, that would benefit from administration of ananti-TCR antibody used, e.g., to stimulate certain population of T cellsin vivo for numeric expansion, for detection of an anti-TCR polypeptide(e.g., for a diagnostic procedure) and/or from treatment, i.e.,palliation or prevention of a disease, with an anti-TCR antibody. Asdescribed in more detail herein, an anti-TCR antibody can be used inunconjugated form or can be conjugated, e.g., to a drug, prodrug, or anisotope.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating certain embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

II. INTRODUCTION

Therapeutics that employ T-cells for targeting specific antigens (e.g.,tumor-associated antigens) are currently being investigated for thetreatment of a variety of disease from cancers to infectious disease.However, the complexity of these agents requires specialized tools forthe purification, propagation and/or activation of T-cells. Embodimentsof the application provide antibodies or antigen binding fragmentsthereof, e.g., scFv, that specifically bind to T-cells, in particularthrough binding to TCR with moderate affinity and can be used toselectively purify, propagate and/or activate T-cells, independent ofthe epitope that is recognized by the T-cell. For example, in oneaspect, antibodies are provided that specifically bind TCRα chain andengages T-cells that recognize a plurality of T-cell epitopes. Themodified 79A-15 (“S15”), 79A-11 (“S11”), 79A-13 (“S13”), 79A-23 (“S23”)monoclonal antibodies, for instance, specifically bind to TCRα withinspecific conformation so as to engage αβ-TCR. This conformationalspecific binding is used to analyze, isolate or propagate and/oractivate T-cells. aAPCs, such as K562 cells, comprising a cell surfaceanti-TCR antibody of the embodiments can be used to activate and therebyto propagate primary T-cell populations (see, e.g., FIG. 7). In someaspects of the invention, K562 cells are HLAC positive, in otheraspects, K562 cells are HLAC negative. Moreover, T-cell populationsexpanded by such aAPC expressing anti-TCR antibodies demonstrate a widevariety of clonotypes encompassing polyclonal T-cell repertoire (FIGS.8-10). Further, optimal stimulation of T cells through a TCR directedscFv preserved donor T cells repertoire better than similar other methodof T cell stimulation currently in vogue. Thus, the methods detailedherein provide new methods for generalized T-cell expansion.

III. TARGET POLYPEPTIDE

In embodiments, the binding molecules of the invention specifically bindto a T-cell receptor containing an α and a β chain (αβ-TCR). Inembodiments, the binding molecules of the invention specifically bind tothe invariant region of the TCRα chain, located at the Va24-J18 junctionregion (GSTLGR (SEQ ID NO:1)) of the T-cell receptor α-chain. Inembodiments, T-cells can be expanded and/or activated by αβ-TCRcross-linking via a TCR alpha chain specific binding molecule in αβ TCR⁺T-cells. In embodiments, the binding molecules of the inventiontherefore specifically bind TCRs containing the invariant GSTLGR (SEQ IDNO:1) regardless of T cell clonotype.

IV. ANTI-TCR ANTIBODIES

In certain embodiments, an antibody or a fragment thereof that binds toTCR polypeptide and stimulates T-cell growth ex vivo. In certainaspects, the antibodies provided here bind to TCR essentiallyindependently of epitope recognized by the TCR and can be used tostimulate expansion of T-cells having a variety of clonotypes. Theantibody may be selected from the group consisting of a chimericantibody, an affinity matured antibody, a polyclonal antibody, amonoclonal antibody, a humanized antibody, a human antibody, or anantigen-binding antibody fragment or a natural or synthetic ligand.Preferably, the anti-TCR antibody is a monoclonal antibody or ahumanized antibody.

Examples of antibody fragments suitable for the present embodimentsinclude, without limitation: (i) the Fab fragment, consisting of V_(L),V_(H), C_(L), and C_(H1) domains; (ii) the “Fd” fragment consisting ofthe V_(H) and C_(H1) domains; (iii) the “Fv” fragment consisting of theV_(L) and V_(H) domains of a single antibody; (iv) the “dAb” fragment,which consists of a V_(H) domain; (v) isolated CDR regions; (vi) F(ab′)2fragments, a bivalent fragment comprising two linked Fab fragments;(vii) single chain Fv molecules (“scFv”), wherein a V_(H) domain and aV_(L) domain are linked by a peptide linker that allows the two domainsto associate to form a binding domain; (viii) bi-specific single chainFv dimers (see U.S. Pat. No. 5,091,513); and (ix) diabodies, multivalentor multispecific fragments constructed by gene fusion (US Patent App.Pub. 20050214860). Fv, scFv, or diabody molecules may be stabilized bythe incorporation of disulphide bridges linking the V_(H) and V_(L)domains. Minibodies comprising a scFv joined to a CH3 domain may also bemade (Hu et al., 1996).

Antibody-like binding peptidomimetics are also contemplated inembodiments. Liu et al. (2003) describe “antibody like bindingpeptidomimetics” (ABiPs), which are peptides that act as pared-downantibodies and have certain advantages of longer serum half-life as wellas less cumbersome synthesis methods.

In one embodiment, the antibody is a chimeric antibody, for example, anantibody comprising antigen binding sequences from a non-human donorgrafted to a heterologous non-human, human, or humanized sequence (e.g.,framework and/or constant domain sequences). Methods have been developedto replace light and heavy chain constant domains of the monoclonalantibody with analogous domains of human origin, leaving the variableregions of the foreign antibody intact. Alternatively, “fully human”monoclonal antibodies are produced in mice transgenic for humanimmunoglobulin genes. Methods have also been developed to convertvariable domains of monoclonal antibodies to more human form byrecombinantly constructing antibody variable domains having both rodent,for example, mouse, and human amino acid sequences. In “humanized”monoclonal antibodies, only the hypervariable CDR is derived from mousemonoclonal antibodies, and the framework and constant regions arederived from human amino acid sequences (see U.S. Pat. Nos. 5,091,513and 6,881,557). It is thought that replacing amino acid sequences in theantibody that are characteristic of rodents with amino acid sequencesfound in the corresponding position of human antibodies will reduce thelikelihood of adverse immune reaction during therapeutic use. Ahybridoma or other cell producing an antibody may also be subject togenetic mutation or other changes, which may or may not alter thebinding specificity of antibodies produced by the hybridoma.

Substitutional variants typically contain the exchange of one amino acidfor another at one or more sites within the protein, and may be designedto modulate one or more properties of the polypeptide, with or withoutthe loss of other functions or properties. Substitutions may beconservative, that is, one amino acid is replaced with one of similarshape and charge. Conservative substitutions are well known in the artand include, for example, the changes of: alanine to serine; arginine tolysine; asparagine to glutamine or histidine; aspartate to glutamate;cysteine to serine; glutamine to asparagine; glutamate to aspartate;glycine to proline; histidine to asparagine or glutamine; isoleucine toleucine or valine; leucine to valine or isoleucine; lysine to arginine;methionine to leucine or isoleucine; phenylalanine to tyrosine, leucineor methionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine. Alternatively, substitutions may benon-conservative such that a function or activity of the polypeptide isaffected. Non-conservative changes typically involve substituting aresidue with one that is chemically dissimilar, such as a polar orcharged amino acid for a nonpolar or uncharged amino acid, and viceversa.

Proteins may be recombinant, or synthesized in vitro. Alternatively, anon-recombinant or recombinant protein may be isolated from bacteria. Itis also contemplated that a bacteria containing such a variant may beimplemented in compositions and methods. Consequently, a protein neednot be isolated.

An antibody or preferably an immunological portion of an antibody, canbe chemically conjugated to, or expressed as, a fusion protein withother proteins. For purposes of this specification and the accompanyingclaims, all such fused proteins are included in the definition ofantibodies or an immunological portion of an antibody.

In embodiments, the invention provides an isolated antibody orantigen-binding fragment thereof that specifically binds to an epitopeof T-cell receptor alpha (TCRα) polypeptide comprising sequence GSTLRG(SEQ ID NO:1).

In embodiments, the antibody or antigen-binding fragment thereof thatspecifically binds to TCRα polypeptide, comprises (a) HCDR1 at least80%, 85%, 90% or 95% identical to SEQ ID NO: 2; (b) HCDR2 at least 80%,85%, 90% or 95% identical to SEQ ID NO: 3; (c) HCDR3 is identical toCDYW (SEQ ID NO:21); CAYW (SEQ ID NO:23); or CAYL (SEQ ID NO:22); (d)LCDR1 at least 80%, 85%, 90% or 95% identical to SEQ ID NO: 4 or SEQ IDNO: 9; (e) LCDR2 at least 80%,85%, 90% or 95% identical to SEQ ID NO: 5;and (f) LCDR3 at least 80%, 85%, 90% or 95% identical to SEQ ID NO: 6 orSEQ ID NO: 10. In embodiments, The antibody or antigen-binding fragmentthereof, comprises (a) HCDR1 identical to SEQ ID NO: 2; (b) HCDR2identical to SEQ ID NO: 3; (c) HCDR3 identical to CDYW (SEQ ID NO:21);CAYW (SEQ ID NO:23); or CAYL (SEQ ID NO:22); (d) LCDR1 identical to SEQID NO: 4; or SEQ ID NO: 9; (e) LCDR2 identical to SEQ ID NO: 5; and (f)LCDR3 identical to SEQ ID NO: 6; or SEQ ID NO: 10.

The invention further provides an antibody or antigen-binding fragmentthereof that specifically binds to TCRα polypeptide comprising (a) HCDR1at least 80%, 85%, 90% or 95% identical to V_(H) CDR1 of 79A-15 SEQ IDNO: 2; (b) HCDR2 at least 80%, 85%, 90% or 95% identical to V_(H) CDR2of 79A-15 SEQ ID NO: 3; (c) HCDR3 at least 80%, 85%, 90% or 95%identical to V_(H) CDR3 of 79A-15 CAYL (SEQ ID NO:22); (d) LCDR1 atleast 80%, 85%, 90% or 95% identical to V_(L) CDR1 of 79A-15 SEQ ID NO:9; (e) LCDR2 at least 80%, 85%, 90% or 95% identical to V_(L) CDR2 of79A-15 SEQ ID NO: 5; and (f) LCDR3 at least 80%, 85%, 90% or 95%identical to V_(L) CDR3 of 79A-15 SEQ ID NO: 10.

The invention additionally provides an isolated antibody or antigenbinding fragment that specifically binds TCRα polypeptide, comprising aV_(H) domain at least about 80%, 85%, 90% or 95% identical to the V_(H)domain of 79A-15 (SEQ ID NO: 11) and a V_(L) domain at least about 80%,85%, 90% or 95% identical to the V_(L) domain of 79A-15 (SEQ ID NO: 12).In embodiments, the isolated antibody or antigen binding fragmentcomprises a V_(L) domain between 90% and 99% identical to the V_(H)domain of 79A-15 (SEQ ID NO: 11) and a VL domain between 90 and 99%identical to the V_(L) domain of 79A-15 (SEQ ID NO: 12). In additionalembodiments, the isolated antibody or antigen binding fragment thereofcomprises a V_(H) domain identical to the V_(H) domain of 79A15 (SEQ IDNO: 11) and a V_(L) domain identical to the V_(L) domain of 79A-15 (SEQID NO: 12).

The invention further provides an isolated antibody or antigen bindingfragment thereof that specifically binds to TCRα polypeptide, comprising(a) HCDR1 at least 80%, 85%, 90% or 95% identical to V_(H) CDR1 of79A-13 KASGYTFTDYYMNWV (SEQ ID NO: 2); (b) HCDR2 at least 80%, 85%, 90%or 95% identical to V_(H) CDR2 of 79A-13 WIGEINPNN (SEQ ID NO: 3); (c)HCDR3 at least 80%, 85%, 90% or 95% identical to V_(H) CDR3 of 79A-13CAYL (SEQ ID NO:22); (d) LCDR1 at least 80%, 85%, 90% or 95% identicalto V_(L) CDR1 of 79A-13 NTYLEWY (SEQ ID NO: 4); (e) LCDR2 at least 80%,85%, 90% or 95% identical to V_(L) CDR2 of 79A-13 KLLIYKVSNRFS (SEQ IDNO: 5); and (f) LCDR3 at least 80%, 85%, 90% or 95% identical to V_(L)CDR3 of 79A-13 MQGSHVPW (SEQ ID NO: 10). In additional embodiments, theinvention provides an isolated antibody or antigen binding fragmentthereof, comprising (a) HCDR1 identical to KASGYTFTDYYMNWV (SEQ ID NO:2); (b) HCDR2 identical to WIGEINPNN (SEQ ID NO: 3); (c) HCDR3 identicalto CAYL (SEQ ID NO:22); (d) LCDR1 identical to NTYLEWY (SEQ ID NO: 4);(e) LCDR2 is identical to KLLIYKVSNRFS (SEQ ID NO: 5); and (f) LCDR3identical to MQGSHVPW (SEQ ID NO: 10).

In additional embodiments, the invention provides isolated antibody orantigen binding fragment thereof that specifically binds to TCRαpolypeptide comprising a V_(H) domain at least about 80%, 85%, 90% or95% identical to the V_(H) domain of 79A-13 (SEQ ID NO: 11) and a V_(L)domain at least about 80%, 85%, 90% or 95% identical to the V_(L) domainof 79A-13 (SEQ ID NO: 14). In embodiments, the isolated antibody orantigen binding fragment comprises a V_(H) domain between 90% and 99%identical to the V_(H) domain of 79A-13 (SEQ ID NO: 11) and a V_(L)domain between 90% and 99% identical to the V_(L) domain of 79A-13 (SEQID NO: 14). In further embodiments, the isolated antibody or antigenbinding fragment comprises a V_(H) domain identical to the V_(H) domainof 79A-13 (SEQ ID NO: 11) and a V_(L) domain identical to the V_(L)domain of 79A-13 (SEQ ID NO: 14).

The invention further provides an antibody or antigen-binding fragmentthereof that specifically binds to TCRα polypeptide, comprising (a)HCDR1 at least 80%, 85%, 90% or 95% identical to V_(H) CDR1 of 79A-11KASGYTFTDYYMNWV (SEQ ID NO: 2); (b) HCDR2 at least 80%, 85%, 90% or 95%identical to V_(H) CDR2 of 79A-11 WIGEINPNN (SEQ ID NO: 3); (c) HCDR3 atleast 80%, 85%, 90% or 95% identical to V_(H) CDR3 of 79A-11 CAYW (SEQID NO:23); (d) LCDR1 at least 80%, 85%, 90% or 95% identical to V_(L)CDR1 of 79A-11 NTYLEWF (SEQ ID NO: 9); (e) LCDR2 at least 80%, 85%, 90%or 95% identical to V_(L) CDR2 of 79A-11 KLLIYKVSNRFS (SEQ ID NO: 5);and (f) LCDR3 at least 80%, 85%, 90% or 95% identical to V_(L) CDR3 of79A-11 MQGSHVPW (SEQ ID NO: 10). In embodiments, the antibody orantigen-binding fragment thereof comprises (a) HCDR1 is identical toKASGYTFTDYYMNWV (SEQ ID NO: 2); (b) HCDR2 is identical to WIGEINPNN (SEQID NO: 3); (c) HCDR3 is identical to CAYW (SEQ ID NO:23); (d) LCDR1 isidentical to NTYLEWF (SEQ ID NO: 9); (e) LCDR2 is identical toKLLIYKVSNRFS (SEQ ID NO: 5); and (f) LCDR3 is identical to MQGSHVPW (SEQID NO: 10).

In embodiments, the invention provides an antibody or antigen-bindingfragment thereof that specifically binds to TCRα polypeptide, comprisinga V_(H) domain at least about 80%, 85%, 90% or 95% identical to theV_(H) domain of 79A-11 (SEQ ID NO: 13) and a V_(L) domain at least about80%, 85%, 90% or 95% identical to the V_(L) domain of 79A-11 (SEQ ID NO:12). In embodiments, the isolated antibody or antigen binding fragmentthereof comprises a V_(H) domain between 90% and 99% identical to theV_(H) domain of 79A-11 (SEQ ID NO: 13) or a V_(L) domain between 90% and99% identical to the V_(L) domain of 79A-11 (SEQ ID NO: 12). Inadditional embodiments, the isolated antibody or antigen bindingfragment thereof of claim 16, comprising a V_(H) domain identical to theV_(H) domain of 79A11 (SEQ ID NO: 13) and a V_(L) domain identical tothe V_(L) domain of 79A11 (SEQ ID NO: 12).

The invention further provides an isolated antibody or antigen bindingfragment thereof that specifically binds to TCRα polypeptide, comprising(a) HCDR1 at least 80%, 85%, 90% or 95% identical to V_(H) CDR1 of 79AKASGYTFTDYYMNWV (SEQ ID NO: 2); (b) HCDR2 at least 80%, 85%, 90% or 95%identical to V_(H) CDR2 of 79A WIGEINPNN (SEQ ID NO: 3); (c) HCDR3 atleast 80%, 85%, 90% or 95% identical to V_(H) CDR3 of 79A CDYW (SEQ IDNO:21); (d) LCDR1 at least 80%, 85%, 90% or 95% identical to V_(L) CDR1of 79A NTYLEWY (SEQ ID NO: 4); (e) LCDR2 at least 80%, 85%, 90% or 95%identical to V_(L) CDR2 of 79A KLLIYKVSNRFS (SEQ ID NO: 5); and (f)LCDR3 at least 80%, 85%, 90% or 95% identical to V_(L) CDR3 of 79AFQGSHVPW (SEQ ID NO: 6). In embodiments, the isolated antibody orantigen binding fragment comprises (a) HCDR1 is identical toKASGYTFTDYYMNWV (SEQ ID NO: 2); (b) HCDR2 is identical to WIGEINPNN (SEQID NO: 3); (c) HCDR3 is identical to CDYW (SEQ ID NO:21); (d) LCDR1 isidentical to NTYLEWY (SEQ ID NO: 4); (e) LCDR2 is identical toKLLIYKVSNRFS (SEQ ID NO: 5); and (f) LCDR3 is identical to FQGSHVPW (SEQID NO: 6).

The invention further provides an isolated antibody or antigen bindingfragment thereof that specifically binds to TCRα polypeptide, comprisinga V_(H) domain at least about 80%, 85%, 90% or 95% identical to theV_(H) domain of 79A (SEQ ID NO: 7) and a V_(L) domain at least about80%, 85%, 90% or 95% identical to the V_(L) domain of 79A (SEQ ID NO:8). In embodiments, the isolated antibody or antigen binding fragmentcomprises a V_(H) domain between 90% and 99% identical to the V_(H)domain of 79A (SEQ ID NO: 7) or a V_(L) domain between 90% and 99%identical to the V_(L) domain of 79A (SEQ ID NO: 8). In embodiments, theisolated antibody or antigen binding fragment comprises a V_(H) domainidentical to the V_(H) domain of 79A (SEQ ID NO: 7) and a V_(L) domainidentical to the V_(L) domain of 79A (SEQ ID NO: 8).

In embodiments, the invention provides an isolated antibody or antigenbinding fragment thereof that specifically binds to TCRα polypeptide,comprising (a) HCDR1 at least 80%, 85%, 90% or 95% identical toKASGYTFTGYYMNWV (SEQ ID NO:15); (b) HCDR2 at least 80%, 85%, 90% or 95%identical to WIGGINPNN (SEQ ID NO:16); (c) HCDR3 identical to CRYW (SEQID NO:17); (d) LCDR1 at least 80%, 85%, 90% or 95% identical toQSIVHGGGNTY (SEQ ID NO:18); (e) LCDR2 at least 80%, 85%, 90% or 95%identical to KLLIYKVSNRFS (SEQ ID NO: 5); and (f) LCDR3 at least 80%,85%, 90% or 95% identical to FQGSHVPW (SEQ ID NO: 6). In embodiments,the isolated antibody or antigen binding fragment thereof comprises (a)HCDR1 identical to KASGYTFTGYYMNWV (SEQ ID NO:15); (b) HCDR2 identicalto WIGGINPNN (SEQ ID NO:16); (c) HCDR3 identical to CRYW (SEQ ID NO:17);(d) LCDR1 identical to QSIVHGGGNTY (SEQ ID NO:18); (e) LCDR2 identicalto KLLIYKVSNRFS (SEQ ID NO: 5); and (f) LCDR3 identical to FQGSHVPW (SEQID NO: 6).

The invention further provides anti-TCR antibody or antigen bindingfragments thereof, comprising a V_(H) domain at least about 80%, 85%,90% or 95% identical to the V_(H) domain of 79A-23 (SEQ ID NO: 19) and aV_(L) domain at least about 80%, 85%, 90% or 95% identical to the V_(L)domain of 79A-23 (SEQ ID NO:20). In embodiments, the isolated antibodyor antigen binding fragment thereof comprises a V_(H) domain between 90%and 99% identical to the V_(H) domain of 79A-23 (SEQ ID NO: 19) and aV_(L) domain between 90 and 99% identical to the V_(L) domain of 79A-23(SEQ ID NO: 20). In further embodiments, the isolated antibody orantigen binding fragment thereof comprises a V_(H) domain identical tothe V_(H) domain of 79A-23 (SEQ ID NO: 19) and a V_(L) domain identicalto the V_(L) domain of 79A-23 (SEQ ID NO: 20).

In embodiments, the anti-TCR antibody or antigen binding fragmentsthereof provided herein are multispecific, e.g., bispecific. Inembodiments, the antibodies and antigen-binding fragments providedherein that specifically bind to TCRα polypeptide are a Fab fragment, aFv fragment and/or are single chain. In embodiments, the anti-TCRantibodies and antigen-binding fragments provided herein are multivalentand comprise at least two heavy chains and at least two light chains. Inembodiments, the antibodies and antigen-binding fragments providedherein that specifically bind to TCRα polypeptide comprise a light chainconstant region selected from the group consisting of a human kappaconstant region and a human lambda constant region.

In embodiments, the anti-TCR antibodies and antigen-binding fragmentsprovided herein comprise a heavy chain constant region or fragmentthereof. In embodiments, the antibodies and antigen-binding fragmentsprovided herein that specifically bind to TCRα polypeptide comprise aheavy chain constant region or fragment thereof which is human IgG1,IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgE, or IgD.

In embodiments, the antibody or antigen binding fragment thereofspecifically binds to an epitope of T-cell receptor alpha (TCRα)polypeptide comprising the sequence GSTLRG (SEQ ID NO:1) with anaffinity characterized by a dissociation constant (KD) no greater than5×10′ M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M,5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M,5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 8.4×10⁻¹² M, 10⁻¹² M,5×10⁻¹³ M , 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M or 10⁻¹⁵ M. Inembodiments, the K_(D) is about 5×10⁻⁹ M to about 6×10⁻⁹M. In furtherembodiments, the K_(D) is about 1×10⁻⁹M to about 2×10⁻⁹M.

In embodiments, the antibodies and antigen-binding fragments providedherein that specifically bind to TCRα polypeptide is humanized,primatized or chimeric. In embodiments, the antibodies andantigen-binding fragments provided herein that specifically bind to TCRαpolypeptide is humanized.

In embodiments, the anti-TCR antibody or antigen binding fragmentthereof of the invention comprises a VH and VL domain separated by alinker. In embodiments the antibody or antigen binding fragment thereofof the invention, further comprises a transmembrane domain. Inembodiments, the antibody or antigen binding fragment thereof comprisinga VH and VL domain separated by a linker comprises a VH polypeptide atleast 90% identical to a polypeptide selected from the group consistingof a SEQ ID NO:11, SEQ ID NO:13 and SEQ ID NO:19, and the VL is apolypeptide at least 90% identical to a polypeptide selected from thegroup consisting of SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:20.

In embodiments, the anti-TCR antibody or fragment disclosed hereincontains a VH polypeptide at least 90% identical to SEQ ID NO:11 and aVL polypeptide at least 90% identical to SEQ ID NO:12, wherein the VHand VL is separated by a linker.

In embodiments, the anti-TCR antibody or fragment disclosed hereincontains a VH polypeptide at least 90% identical to SEQ ID NO:13 and aVL polypeptide at least 90% identical to SEQ ID NO:12, wherein the VHand VL is separated by a linker.

The invention further provides an anti-TCR antibody or fragmentcontaining a VH polypeptide at least 90% identical to SEQ ID NO:11 and aVL polypeptide at least 90% identical to SEQ ID NO:14, wherein the VHand VL is separated by a linker.

In embodiments, the anti-TCR antibody or fragment disclosed hereincontains a VH polypeptide at least 90% identical to SEQ ID NO:19 and aVL polypeptide at least 90% identical to SEQ ID NO:20, wherein the VHand VL is separated by a linker.

Thus, in embodiments, the anti-TCR antibody or antigen-binding fragmentdisclosed herein is a single-chain Fv fragment (scFv), e.g., aheterodimer containing the VH and VL domains, which are connected by alinker, forming a single polypeptide.

In embodiments, the linker is a polypeptide between 10-30 amino acids inlength, between 15-25 amino acids in length, between 15 and 20 aminoacids in length. In embodiments, the linker is 15, 16, 17, 18, 19, 20,21, 22, 23, or 25 amino acids in length. Suitable linkers for scFv areknown to those of skill in the art. For example, multimers of the GGGGS(G45 or Gly4Ser) are suitable linkers. Exemplary G45 multimers includethe 15-mer (G4S)₃, and the 20-mers (G4s)₄. A further exemplary linker isthe 18-mer GGSSRSSSSGGGGSGGGG. Exemplary sequences with addedfunctionalities, such as an epitope tag or an encoding sequencecontaining a Cre-Lox recombination site or sequences improving scFvproperties, are also contemplated as linker sequences.

V. POLYNUCLEOTIDES ENCODING ANTI-TCR ANTIBODIES

The present invention also includes fragments of the polynucleotides ofthe invention, as described elsewhere. Additionally, polynucleotidesthat encode fusion polypeptides, Fab fragments, and other derivatives,as described herein, are also contemplated by the invention.

The polynucleotides are produced or manufactured by methods known in theart. For example, if the nucleotide sequence of the antibody is known, apolynucleotide encoding the antibody may be assembled from chemicallysynthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis ofoverlapping oligonucleotides containing portions of the sequenceencoding the antibody, annealing and ligating of those oligonucleotides,and then amplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an anti-TCR antibody, orantigen-binding fragment, variant, or derivative thereof of theinvention, may be generated from nucleic acid from a suitable source. Ifa clone containing a nucleic acid encoding a particular antibody is notavailable, but the sequence of the antibody molecule is known, a nucleicacid encoding the antibody may be chemically synthesized or obtainedfrom a suitable source (e.g., an antibody cDNA library, or a cDNAlibrary generated from, or nucleic acid, preferably poly A+RNA, isolatedfrom, any tissue or cells expressing the antibody or other anti-TCRantibody, such as hybridoma cells selected to express an antibody) byPCR amplification using synthetic primers hybridizable to the 3′ and 5′ends of the sequence or by cloning using an oligonucleotide probespecific for the particular gene sequence to identify, e.g., a cDNAclone from a cDNA library that encodes the antibody or other anti-TCRantibody. Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe anti-TCR antibody, or antigen-binding fragment, variant, orderivative thereof is determined, its nucleotide sequence may bemanipulated using methods well known in the art for the manipulation ofnucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al. (1990) Molecular Cloning, A Laboratory Manual (2nd ed.;Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) and Ausubel etal., eds. (1998) Current Protocols in Molecular Biology (John Wiley &Sons, NY), which are both incorporated by reference herein in theirentireties), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

A polynucleotide encoding an anti-TCR binding molecule, e.g., anantibody, or antigen-binding fragment, variant, or derivative thereof,can be composed of any polyribonucleotide or polydeoxyribonucleotide,which may be unmodified RNA or DNA or modified RNA or DNA. For example,a polynucleotide encoding anti-TCR antibody, or antigen-bindingfragment, variant, or derivative thereof can be composed of single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, a polynucleotide encoding an anti-TCR binding molecule, e.g.,an antibody, or antigen-binding fragment, variant, or derivative thereofcan be composed of triple-stranded regions comprising RNA or DNA or bothRNA and DNA. A polynucleotide encoding an anti-TCR binding molecule,e.g., antibody, or antigen-binding fragment, variant, or derivativethereof, may also contain one or more modified bases or DNA or RNAbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

An isolated polynucleotide encoding a non-natural variant of apolypeptide derived from an immunoglobulin (e.g., an immunoglobulinheavy chain portion or light chain portion) can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of the immunoglobulin such that one or moreamino acid substitutions, additions or deletions are introduced into theencoded protein. Mutations may be introduced by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.Preferably, conservative amino acid substitutions are made at one ormore non-essential amino acid residues.

In embodiments, the invention provides an isolated polynucleotidecomprising a nucleic acid encoding an antibody or antigen bindingfragment thereof that specifically binds to a TCRα chain, said antibodybinding to an epitope of TCRα polypeptide comprising the sequence GSTLRG(SEQ ID NO: 1).

In embodiments, the invention provides a nucleic acid encoding a VHpolypeptide at least 90% identical to SEQ ID NO:11, SEQ ID NO:13 or SEQID NO:19. In embodiments, the invention provides a nucleic acid encodinga nucleic acid encoding a VL polypeptide at least 90% identical to, SEQID NO:12, SEQ ID NO:14 or SEQ ID NO:20.

The invention further provides a nucleic acid encoding a HCDR1 aminoacid sequence identical to SEQ ID NO:2 or SEQ ID NO:15; a HCDR2 aminoacid sequence identical to SEQ ID NO:3 or SEQ ID NO:16; and/or a HCDR3amino acid sequence identical to SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, or SEQ ID NO:17.

The invention further provides a nucleic acid encoding a LCDR1 aminoacid sequence identical to SEQ ID NO:4, 9 or 18; a LCDR2 amino acidsequence identical to SEQ ID NO:5, and/or a nucleic acid encoding aLCDR3 amino acid sequence identical to SEQ ID NO:6, or 10.

The invention further provides an isolated polynucleotide comprising anucleic acid encoding a VH polypeptide, wherein said VH polypeptidecomprises HCDR1, HCDR2 and HCDR3 amino acid sequences comprising SEQ IDNOs:2, 3, and 21, respectively, and wherein an antibody or antigenfragment comprising said VH polypeptide specifically binds an epitope ofT-cell receptor alpha (TCRα) polypeptide comprising sequence GSTLRG (SEQID NO:1).

The invention also provides an isolated polynucleotide comprising anucleic acid encoding a VH polypeptide, wherein said VH polypeptidecomprises HCDR1, HCDR2, and HCDR3 amino acid sequences comprising SEQ IDNOs: 2, 3, and 22, respectively, and wherein an antibody or antigenfragment comprising said VH polypeptide specifically binds an epitope ofT-cell receptor alpha (TCRα) polypeptide comprising sequence GSTLRG (SEQID NO:1).

In embodiments, the invention is directed to an isolated polynucleotidecomprising a nucleic acid encoding a VH polypeptide, wherein said VHpolypeptide comprises HCDR1, HCDR2, and HCDR3 amino acid sequencescomprising SEQ ID NOs: 2, 3, and 23, respectively, and wherein anantibody or antigen fragment comprising said VH polypeptide specificallybinds an epitope of T-cell receptor alpha (TCRα) polypeptide comprisingsequence GSTLRG (SEQ ID NO:1).

In further embodiments, the invention provides an isolatedpolynucleotide comprising a nucleic acid encoding a VH polypeptide,wherein said VH polypeptide comprises HCDR1, HCDR2, and HCDR3 amino acidsequences comprising SEQ ID NOs:15, 16, and 17, respectively, andwherein an antibody or antigen fragment comprising said VH polypeptidespecifically binds an epitope of T-cell receptor alpha (TCRα)polypeptide comprising sequence GSTLRG (SEQ ID NO:1).

The invention is further directed to, in embodiments, an isolatedpolynucleotide comprising a nucleic acid encoding a VL polypeptide,wherein said VL polypeptide comprises LCDR1, LCDR2, and LCDR3 amino acidsequences comprising SEQ ID NOs:4, 5 and 6, respectively, and wherein anantibody or antigen fragment comprising said VL polypeptide specificallybinds an epitope of T-cell receptor alpha (TCRα) polypeptide comprisingsequence GSTLRG (SEQ ID NO:1).

In embodiments, the invention provides an isolated polynucleotidecomprising a nucleic acid encoding a VL polypeptide, wherein said VLpolypeptide comprises LCDR1, LCDR2, and LCDR3 amino acid sequencescomprising SEQ ID NOs: 9, 5 and 10, respectively, and wherein anantibody or antigen fragment comprising said VL polypeptide specificallybinds an epitope of T-cell receptor alpha (TCRα) polypeptide comprisingsequence GSTLRG (SEQ ID NO:1).

The invention further provides an isolated polynucleotide comprising anucleic acid encoding a VL polypeptide, wherein said VL polypeptidecomprises LCDR1, LCDR2, and LCDR3 amino acid sequences comprising SEQ IDNOs: 4, 5 and 10, respectively, and wherein an antibody or antigenfragment comprising said VL polypeptide specifically binds an epitope ofT-cell receptor alpha (TCRα) polypeptide comprising sequence GSTLRG (SEQID NO:1).

In embodiments, the invention provides an isolated polynucleotidecomprising a nucleic acid encoding a VL polypeptide, wherein said VLpolypeptide comprises LCDR1, LCDR2, and LCDR3 amino acid sequencescomprising SEQ ID NOs: 18, 5 and 6, respectively, and wherein anantibody or antigen fragment comprising said VL polypeptide specificallybinds an epitope of T-cell receptor alpha (TCRα) polypeptide comprisingsequence GSTLRG (SEQ ID NO:1).

VI. FUSION PROTEINS AND ANTIBODY CONJUGATES

As discussed in more detail elsewhere herein, anti-TCR bindingmolecules, e.g., antibodies of the invention, or antigen-bindingfragments, variants, or derivatives thereof, may further berecombinantly fused to a heterologous polypeptide at the N- orC-terminus or chemically conjugated (including covalent and non-covalentconjugations) to polypeptides or other compositions. For example,anti-TCR antibodies may be recombinantly fused or conjugated tomolecules useful as labels in detection assays and effector moleculessuch as heterologous polypeptides, drugs, radionuclides, or toxins. See,e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat.No. 5,314,995; and EP 396,387.

Anti-TCR antibodies of the invention, or antigen-binding fragments,variants, or derivatives thereof, may include derivatives that aremodified, i.e., by the covalent attachment of any type of molecule tothe antibody such that covalent attachment does not prevent the antibodyfrom binding to the target TCR. For example, but not by way oflimitation, the antibody derivatives include antibodies that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, etc. Additionally, the derivativemay contain one or more non-classical amino acids.

Anti-TCR binding molecules, e.g., antibodies of the invention, orantigen-binding fragments, variants, or derivatives thereof, can becomposed of amino acids joined to each other by peptide bonds ormodified peptide bonds, i.e., peptide isosteres, and may contain aminoacids other than the 20 gene-encoded amino acids. For example, anti-TCRantibodies may be modified by natural processes, such asposttranslational processing, or by chemical modification techniquesthat are well known in the art. Such modifications are well described inbasic texts and in more detailed monographs, as well as in a voluminousresearch literature. Modifications can occur anywhere in the anti-TCRbinding molecule, including the peptide backbone, the amino acidside-chains and the amino or carboxyl termini, or on moieties such ascarbohydrates. It will be appreciated that the same type of modificationmay be present in the same or varying degrees at several sites in agiven anti-TCR binding molecule. Also, a given anti-TCR binding moleculemay contain many types of modifications. Anti-TCR binding molecules maybe branched, for example, as a result of ubiquitination, and they may becyclic, with or without branching. Cyclic, branched, and branched cyclicanti-TCR binding molecule may result from posttranslation naturalprocesses or may be made by synthetic methods. Modifications includeacetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, pegylation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins such asarginylation, and ubiquitination, (See, for instance,Proteins--Structure and Molecular Properties, T. E. Creighton, W. H.Freeman and Company, NY; 2nd ed. (1993): Johnson, ed. (1983)Posttranslational Covalent Modification of Proteins (Academic Press,NY), pgs. 1-12; Seifter et al., Meth. Enzymol. 182:626-646 (1990);Rattan et al., Ann. NY Acad. Sci. 663:48-62 (1992)).

The present invention also provides for fusion proteins comprising ananti-TCR antibody, or antigen-binding fragment, variant, or derivativethereof, and a heterologous polypeptide. The heterologous polypeptide towhich the antibody is fused is useful for function or is useful totarget the anti-TCR polypeptide expressing cells. For example, thebinding and/or crosslinking of the TCR leads to T-cell effectorfunctions, such as cytokine production, proliferation and killing. Inanother embodiment, antibody or its derivative including mutants may befused to transmembrane domains of human immune cell surface receptorsfor desirable surface expression of the fusion construct on transfectedcells(s) and/or for enabling desirable target recognition by the chimeracausing improved effector functions.

In one embodiment, a fusion protein of the invention comprises, consistsessentially of, or consists of, a polypeptide having the amino acidsequence of any one or more of the VH domains of an antibody of theinvention or the amino acid sequence of any one or more of the VL,domains of an antibody of the invention or fragments or variantsthereof, and a heterologous polypeptide sequence.

In another embodiment, a fusion protein for use in the diagnostic andtreatment methods disclosed herein comprises, consists essentially of,or consists of a polypeptide having the amino acid sequence of any one,two, three of the CDRs of the VH domain of an anti-TCR antibody, orfragments, variants, or derivatives thereof, or the amino acid sequenceof any one, two, three of the CDRs of the VL domain an anti-TCRantibody, or fragments, variants, or derivatives thereof, and aheterologous polypeptide sequence. In one embodiment, a fusion proteincomprises a polypeptide having the amino acid sequence of at least oneVH domain of an anti-TCR antibody of the invention and the amino acidsequence of at least one VL domain of an anti-TCR antibody of theinvention or fragments, derivatives or variants thereof, and aheterologous polypeptide sequence. Preferably, the VH and VL domains ofthe fusion protein correspond to a single source antibody (or scFv orFab fragment) that specifically binds at least one epitope of TCR. Inyet another embodiment, a fusion protein for use in the diagnostic andtreatment methods disclosed herein comprises a polypeptide having theamino acid sequence of any one, two, three or more of the CDRs of the VHdomain of an anti-TCR antibody and the amino acid sequence of any one,two, three or more of the CDRs of the VL domain of an anti-TCR antibody,or fragments or variants thereof, and a heterologous polypeptidesequence. Preferably, two, three, four, five, six, or more of the CDR(s)of the VH domain or VL domain correspond to single source antibody (orsay or Fab fragment) of the invention. Nucleic acid molecules encodingthese fusion proteins are also encompassed by the invention.

Exemplary fusion proteins reported in the literature include fusions ofthe T cell receptor (Gaseoigne et al., Proc. Natl. Acad. Sci. USA84:2936-2940 (1987)); CD4 (Capon et al., Nature 337:525-531 (1989);Traunecker et al., Nature 339:68-70 (1989); Zettmeissl et al., DNA CellBiol. USA 9:347-353 (1990); and Byrn et al., Nature 344:667-670 (1990));L-selectin (homing receptor) (Watson et al., J. Cell. Biol.110:2221-2229 (1990); and Watson et al., Nature 349:164-167 (1991));CD44 (Aruffo et al., Cell 61:1303-1313 (1990)); CD28 and 137 (Linsley etal., J. Exp. Med. 173:721-730 (1991)); CTLA-4 (Lisley et al., J. Exp.Med. 174:561-569 (1991)); CD22 (Stamenkovie et al., Cell 66:1133-1144(1991)); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA88:10535-10539 (1991); Lesslauer et al., Eur. J. Immunol. 27:2883-2886(1991); and Peppel et al., J. Exp. Med. 174:1483-1489 (1991)); and IgEreceptor a (Ridgway and Gorman, J. Cell. Biol. Vol. 115, Abstract No.1448 (1991)).

As discussed elsewhere herein, anti-TCR binding molecules, e.g.,antibodies of the invention, or antigen-binding fragments, variants, orderivatives thereof, may be fused heterologous polypeptides to increasethe in vivo half-life of the polypeptides or for use in immunoassaysusing methods known in the art. For example, in one embodiment, PEG canbe conjugated to the anti-TCR antibodies of the invention to increasetheir half-life in vivo. See Leong et al., Cytokine 16:106 (2001); Adv.in Drug Deliv. Rev. 54:531 (2002); or Weir et al., Biochem. Soc.Transactions 30:512 (2002).

Moreover, anti-TCR binding molecules, e.g., antibodies of the invention,or antigen-binding fragments, variants, or derivatives thereof, can befused to marker sequences, such as a peptide to facilitate theirpurification or detection. In preferred embodiments, the marker aminoacid sequence is a hexa-histidine peptide, such as the tag provided in apQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311),among others, many of which are commercially available. As described inGentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), forinstance, hexa-histidine provides for convenient purification of thefusion protein. Other peptide tags useful for purification include, butare not limited to, the “HA” tag, which corresponds to an epitopederived from the influenza hemagglutinin protein (Wilson et al., Cell37:767 (1984)) and the “flag” tag.

Fusion proteins can be prepared using methods that are well known in theart (see for example U.S. Pat. Nos. 5,116,964 and 5,225,538). Theprecise site at which the fusion is made may be selected empirically tooptimize the secretion or binding characteristics of the fusion protein.DNA encoding the fusion protein is then transfected into a host cell forexpression.

Anti-TCR binding molecules, e.g., antibodies of the present invention,or antigen-binding fragments, variants, or derivatives thereof, may beused in non-conjugated form or may be conjugated to at least one of avariety of molecules, e.g., to improve the therapeutic properties of themolecule, to facilitate target detection, or for imaging or therapy ofthe patient. Anti-TCR binding molecules, e.g., antibodies of theinvention, or antigen-binding fragments, variants, or derivativesthereof, can be labeled or conjugated either before or afterpurification, or when purification is performed.

In particular, anti-TCR antibodies of the invention, or antigen-bindingfragments, variants, or derivatives thereof, may be conjugated totherapeutic agents, prodrugs, peptides, proteins, enzymes, viruses,lipids, biological response modifiers, pharmaceutical agents, or PEG.

Those skilled in the art will appreciate that conjugates may also beassembled using a variety of techniques depending on the selected agentto be conjugated. For example, conjugates with biotin are prepared,e.g., by reacting a binding polypeptide with an activated ester ofbiotin such as the biotin N-hydroxysuccinimide ester. Similarly,conjugates with a fluorescent marker may be prepared in the presence ofa coupling agent, e.g., those listed herein, or by reaction with anisothiocyanate, preferably fluorescein-isothiocyanate. Conjugates of theanti-TCR antibodies of the invention, or antigen-binding fragments,variants, or derivatives thereof, are prepared in an analogous manner.

The present invention further encompasses anti-TCR binding molecules,e.g., antibodies of the invention, or antigen-binding fragments,variants, or derivatives thereof, conjugated to a diagnostic ortherapeutic agent. The anti-TCR antibodies, including antigen-bindingfragments, variants, and derivatives thereof, can be used diagnosticallyto, for example, monitor the development or progression of a disease aspart of a clinical testing procedure to, e.g., determine the efficacy ofa given treatment and/or prevention regimen. For example, detection canbe facilitated by coupling the anti-TCR antibody, or antigen-bindingfragment, variant, or derivative thereof, to a detectable substance.Examples of detectable substances include various enzymes, prostheticgroups, fluorescent materials, luminescent materials, bioluminescentmaterials, radioactive materials, positron emitting metals using variouspositron emission topographies, and nonradioactive paramagnetic metalions. Sec, for example, U.S. Pat. No. 4,741,900 for metal ions which canbe conjugated to antibodies for use as diagnostics according to thepresent invention. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, .beta.-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ¹¹¹In, ⁹⁰Y, or ⁹⁹Tc.

An anti-TCR binding molecule, e.g., an antibody, or antigen-bindingfragment, variant, or derivative thereof, may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells.

An anti-TCR binding molecule, e.g., an antibody, or antigen-bindingfragment, variant, or derivative thereof, also can be detectably labeledby coupling it to a reporter, such as a chemiluminescent or fluorescentcompound. The presence of the tagged anti-TCR binding molecule is thendetermined by detecting the presence of luminescence that arises duringthe course of a chemical reaction. Examples of particularly usefulchemiluminescent labeling compounds are luminol, isoluminol, theromaticacridinium ester, imidazole, acridinium salt and oxalate ester.

One of the ways in which an anti-TCR antibody, or antigen-bindingfragment, variant, or derivative thereof, can be detectably labeled isby linking the same to an enzyme and using the linked product in anenzyme immunoassay (EIA) (Volley, A., “The Enzyme Linked ImmunosorbentAssay (ELISA)” Microbiological Associates Quarterly Publication,Walkersville, Md.; Diagnostic Horizons 2:1-7 (1978); Voller et al., J.Clin. Pathol. 31:507-520 (1978); Butler, Meth. Enzymol. 73:482-523(1981); Maggio, ed. (1980) Enzyme Immunoassay, CRC Press, Boca Raton,Fla.; Ishikawa et al., eds. (1981) Enzyme Immunoassay (Kgaku Shoin,Tokyo). The enzyme, which is bound to the anti-TCR antibody will reactwith an appropriate substrate, preferably a chromogenic substrate, insuch a manner as to produce a chemical moiety which can be detected, forexample, by spectrophotometric, fluorimetric or by visual means. Enzymeswhich can be used to detectably label the antibody include, but are notlimited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. Additionally, the detection can be accomplished bycolorimetric methods which employ a chromogenic substrate for theenzyme. Detection may also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the anti-TCRbinding molecule, e.g., antibody, or antigen-binding fragment, variant,or derivative thereof, it is possible to detect the binding moleculethrough the use of a radioimmunoassay (MA) (see, for example, Weintraub(March, 1986) Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques (The Endocrine Society), which isincorporated by reference herein). The radioactive isotope can bedetected by means including, but not limited to, a gamma counter, ascintillation counter, or autoradiography.

An anti-TCR binding molecule, e.g., antibody, or antigen-bindingfragment, variant, or derivative thereof, can also be detectably labeledusing fluorescence emitting metals such as 152Eu, or others of thelanthanide series. These metals can be attached to the binding moleculeusing such metal chelating groups as diethylenetriaminepentacetic acid(DTPA) or ethylenediaminetetraacetic acid (EDTA).

Techniques for conjugating various moieties to an antibody (e.g., ananti-TCR antibody), or antigen-binding fragment, variant, or derivativethereof, are well known, see, e.g., Amon et al. (1985) “MonoclonalAntibodies for Immunotargeting of Drugs in Cancer Therapy,” inMonoclonal Antibodies and Cancer Therapy, ed. Reisfeld et al. (Alan R.Liss, Inc.), pp. 243-56; Hellstrom et al. (1987) “Antibodies for DrugDelivery,” in Controlled Drug Delivery, ed. Robinson et al, (2nd ed.;Marcel Dekker, Inc.), pp. 623-53); Thorpe (1985) “Antibody Carriers ofCytotoxic Agents in Cancer Therapy: A Review,” in Monoclonal Antibodies'84: Biological and Clinical Applications, ed. Pinchera et al., pp.475-506; “Analysis, Results, and Future Prospective of the TherapeuticUse of Radiolabeled Antibody in Cancer Therapy,” in MonoclonalAntibodies for Cancer Detection and Therapy, ed. Baldwin et al.,Academic Press, pp. 303-16 (1985); and Thorpe et al. (1982) “ThePreparation and Cytotoxic Properties of Antibody-Toxin Conjug” Immunol.Rev. 62:119-58.

VII. EXPRESSING ANTI-TCR ANTIBODY POLYPEPTIDES

DNA sequences that encode the light and the heavy chains of the antibodymay be made, either simultaneously or separately, using reversetranscriptase and DNA polymerase in accordance with well-known methods.PCR may be initiated by consensus constant region primers or by morespecific primers based on the published heavy and light chain DNA andamino acid sequences. As discussed above, PCR also may be used toisolate DNA clones encoding the antibody light and heavy chains. In thiscase the libraries may be screened by consensus primers or largerhomologous probes, such as mouse constant region probes.

DNA, typically plasmid DNA, may be isolated from cells using techniquesknown in the art, restriction mapped and sequenced in accordance withstandard, well known techniques set forth in detail, e.g., in theforegoing references relating to recombinant DNA techniques. Of course,the DNA may be synthetic according to the present invention at any pointduring the isolation process or subsequent analysis.

Following manipulation of the isolated genetic material to provideanti-TCR antibodies, or antigen-binding fragments, variants, orderivatives thereof, of the invention, the polynucleotides encoding theanti-TCR antibodies are typically inserted in an expression vector forintroduction into host cells that may be used to produce the desiredquantity of anti-TCR antibody.

Recombinant expression of an antibody, or fragment, derivative or analogthereof, e.g., a heavy or light chain of an antibody that binds to atarget molecule described herein, e.g., TCR, requires construction of anexpression vector containing a polynucleotide that encodes the antibody.Once a polynucleotide encoding an antibody molecule or a heavy or lightchain of an antibody, or portion thereof (preferably containing theheavy or light chain variable domain), of the invention has beenobtained, the vector for the production of the antibody molecule may beproduced by recombinant DNA technology using techniques well known inthe art. Thus, methods for preparing a protein by expressing apolynucleotide containing an antibody encoding nucleotide sequence aredescribed herein. Methods that are well known to those skilled in theart can be used to construct expression vectors containing antibodycoding sequences and appropriate transcriptional and translationalcontrol signals. These methods include, for example, in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. The invention, thus, provides replicable vectorscomprising a nucleotide sequence encoding an antibody molecule of theinvention, or a heavy or light chain thereof, or a heavy or light chainvariable domain, operably linked to a promoter. Such vectors may includethe nucleotide sequence encoding the constant region of the antibodymolecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of theantibody may be cloned into such a vector for expression of the entireheavy or light chain.

The term “vector” or “expression vector” is used herein to mean vectorsused in accordance with the present invention as a vehicle forintroducing into and expressing a desired gene in a host cell. As knownto those skilled in the art, such vectors may easily be selected fromthe group consisting of plasmids, phages, viruses and retroviruses. Ingeneral, vectors compatible with the instant invention will comprise aselection marker, appropriate restriction sites to facilitate cloning ofthe desired gene and the ability to enter and/or replicate in eukaryoticor prokaryotic cells.

In particular aspects, the invention provides vectors comprising apolynucleotide encoding the anti-TCR antibodies and antigen bindingfragments as disclosed herein. In embodiments, the vectors are viral,non-viral, episomal, or integrating. In embodiments, the vectors aretransposons, e.g., sleeping beauty transposons. In embodiments, thevector is a vector comprising lentiviral backbone components. Exemplarybackbone components include, but are not limited to, pFUGW, and pSMPUW.

For the purposes of this invention, numerous expression vector systemsmay be employed. For example, one class of vector utilizes DNA elementsthat are derived from animal viruses such as bovine papilloma virus,polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses(RSV, MMTV or MOMLV) or SV40 virus. Others involve the use ofpolycistronic systems with internal ribosome binding sites.Additionally, cells that have integrated the DNA into their chromosomesmay be selected by introducing one or more markers which allow selectionof transfected host cells. The marker may provide for prototrophy to anauxotrophic host, biocide resistance (e.g., antibiotics) or resistanceto heavy metals such as copper. The selectable marker gene can either bedirectly linked to the DNA sequences to be expressed, or introduced intothe same cell by cotransformation. Additional elements may also beneeded for optimal synthesis of mRNA. These elements may include signalsequences, splice signals, as well as transcriptional promoters,enhancers, and termination signals.

In particularly preferred embodiments the cloned variable region genesare inserted into an expression vector along with the heavy and lightchain constant region genes (preferably human) synthesized as discussedabove. Of course, any expression vector that is capable of elicitingexpression in eukaryotic cells may be used in the present invention.Examples of suitable vectors include, but are not limited to plasmidspcDNA3, pHCMV/Zeo, pCR3.1, pEF 1/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2,pTRACER-HCMV, pUB6/V5-His, pVAX1, and pZeoSV2 (available fromInvitrogen, San Diego, Calif.), and plasmid pCI (available from Promega,Madison, Wis.). In general, screening large numbers of transformed cellsfor those that express suitably high levels if immunoglobulin heavy andlight chains is routine experimentation that can be carried out, forexample, by robotic systems.

More generally, once the vector or DNA sequence encoding a monomericsubunit of the anti-TCR antibody has been prepared, the expressionvector may be introduced into an appropriate host cell. Introduction ofthe plasmid into the host cell can be accomplished by various techniqueswell known to those of skill in the art. These include, but are notlimited to, transfection (including electrophoresis andelectroporation), protoplast fusion, calcium phosphate precipitation,cell fusion with enveloped DNA, microinjection, and infection withintact virus. See, Ridgway (1988) “Mammalian Expression Vectors” inVectors, ed. Rodriguez and Denhardt (Butterworths, Boston, Mass.),Chapter 24.2, pp. 470-472. Typically, plasmid introduction into the hostis via electroporation. The host cells harboring the expressionconstruct are grown under conditions appropriate to the production ofthe light chains and heavy chains, and assayed for heavy and/or lightchain protein synthesis. Exemplary assay techniques includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), orfluorescence-activated cell sorter analysis (FACS), immunohistochemistryand the like.

The expression vector is transferred to a host cell by conventionaltechniques, and the transfected cells are then cultured by conventionaltechniques to produce an antibody for use in the methods describedherein. Thus, the invention includes host cells containing apolynucleotide encoding an antibody of the invention, or a heavy orlight chain thereof, operably linked to a heterologous promoter. Inpreferred embodiments for the expression of double-chained antibodies,vectors encoding both the heavy and light chains may be co-expressed inthe host cell for expression of the entire immunoglobulin molecule, asdetailed below.

“Host cells” refers to cells that harbor vectors constructed usingrecombinant DNA techniques and encoding at least one heterologous gene.In descriptions of processes for isolation of antibodies fromrecombinant hosts, the terms “cell” and “cell culture” are usedinterchangeably to denote the source of antibody unless it is clearlyspecified otherwise. In other words, recovery of polypeptide from the“cells” may mean either from spun down whole cells, or from the cellculture containing both the medium and the suspended cells.

A variety of host-expression vector systems may be utilized to expressantibody molecules for use in the methods described herein. Suchhost-expression systems represent vehicles by which the coding sequencesof interest may be produced and subsequently purified, but alsorepresent cells that may, when transformed or transfected with theappropriate nucleotide coding sequences, express an antibody molecule ofthe invention in situ. These include, but are not limited to,microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformedwith recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors containing antibody coding sequences; yeast (e.g.,Saccharomyces, Pichia) transformed with recombinant yeast expressionvectors containing antibody coding sequences; insect cell systemsinfected with recombinant virus expression vectors (e.g., baculovirus)containing antibody coding sequences; plant cell systems infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing antibody codingsequences; or mammalian cell systems (e.g., COS, CHO, BLK, 293, 3T3cells) harboring recombinant expression constructs containing promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the adenovirus late promoter;the vaccinia virus 7.5K promoter). Preferably, bacterial cells such asEscherichia coli, and more preferably, eukaryotic cells, especially forthe expression of whole recombinant antibody molecule, are used for theexpression of a recombinant antibody molecule. For example, mammaliancells such as Chinese hamster ovary cells (CHO), in conjunction with avector such as the major intermediate early gene promoter element fromhuman cytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

The host cell line used for protein expression is often of mammalianorigin; those skilled in the art are credited with ability topreferentially determine particular host cell lines that are best suitedfor the desired gene product to be expressed therein. Exemplary hostcell lines include, but are not limited to, CHO (Chinese Hamster Ovary),DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus), HELA (humancervical carcinoma), CVI (monkey kidney line), COS (a derivative of CVIwith SV40 T antigen), VERY, BHK (baby hamster kidney), MDCK, 293, WI38,R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK(hamster kidney line), SP2/O (mouse myeloma), P3.times.63-Ag3.653 (mousemyeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocyte)and 293 (human kidney). Host cell lines are typically available fromcommercial services, the American Tissue Culture Collection or frompublished literature.

In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells that possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably express theantibody molecule may be engineered. Rather than using expressionvectors that contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which stably express theantibody molecule.

A number of selection systems may be used, including, but not limitedto, the herpes simplex virus thymidine kinase (Wigler et al., Cell11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase(Szybalska and Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), andadenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980))genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshey, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); TIB TECH 11(5):155-215 (May, 1993); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984).Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (1993) Current Protocols inMolecular Biology (John Wiley & Sons, NY); Kriegier (1990) “GeneTransfer and Expression” in A Laboratory Manual (Stockton Press, NY);Dracopoli et al. (eds) (1994) Current Protocols in Human Genetics (JohnWiley & Sons, NY) Chapters 12 and 13; Colberre-Garapin et al. (1981) J.Mol. Biol. 150:1, which are incorporated by reference herein in theirentireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel (1987) “TheUse of Vectors Based on Gene Amplification for the Expression of ClonedGenes in Mammalian Cells in DNA Cloning” (Academic Press, NY) Vol. 3.When a marker in the vector system expressing antibody is amplifiable,increase in the level of inhibitor present in culture of host cell willincrease the number of copies of the marker gene. Since the amplifiedregion is associated with the antibody gene, production of the antibodywill also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).

In vitro production allows scale-up to give large amounts of the desiredpolypeptides. Techniques for mammalian cell cultivation under tissueculture conditions are known in the art and include homogeneoussuspension culture, e.g. in an airlift reactor or in a continuousstirrer reactor, or immobilized or entrapped cell culture, e.g. inhollow fibers, microcapsules, on agarose microbeads or ceramiccartridges. If necessary and/or desired, the solutions of polypeptidescan be purified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose or (immuno-)affinity chromatography, e.g., afterpreferential biosynthesis of a synthetic hinge region polypeptide orprior to or subsequent to the HIC chromatography step described herein.

Genes encoding anti-TCR antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention can also be expressedin non-mammalian cells such as insect, bacteria or yeast or plant cells.Bacteria that readily take up nucleic acids include members of theenterobacteriaceae, such as strains of Escherichia coli or Salmonella;Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, andHaemophilus influenzae. It will further be appreciated that, whenexpressed in bacteria, the heterologous polypeptides typically becomepart of inclusion bodies. The heterologous polypeptides must beisolated, purified and then assembled into functional molecules. Wheretetravalent forms of antibodies are desired, the subunits will thenself-assemble into tetravalent antibodies (WO 02/096948A2).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lacZ coding region sothat a fusion protein is produced; ON vectors (Inouye and Inouye,Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke and Schuster, J. Biol.Chem. 24:5503-5509 (1989)); and the like. pGEX vectors may also be usedto express foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding to amatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In addition to prokaryotes, eukaryotic microbes may also be used.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among eukaryotic microorganisms although a number of other strainsare commonly available, e.g., Pichia pastoris.

For expression in Saccharomyces, the plasmid YRp7, for example,(Stinchcomb et al., Nature 282:39 (1979); Kingsman et al., Gene 7:141(1979); Tschemper et al., Gene 10:157 (1980)) is commonly used. Thisplasmid already contains the TRP1 gene, which provides a selectionmarker for a mutant strain of yeast lacking the ability to grow intryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics 85:12(1977)). The presence of the trp1 lesion as a characteristic of theyeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is typically used as a vector to express foreign genes. Thevirus grows in Spodoptera frugiperda cells. The antibody coding sequencemay be cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

Once an antibody molecule of the invention has been recombinantlyexpressed, it may be purified by any method known in the art forpurification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins.Alternatively, a preferred method for increasing the affinity ofantibodies of the invention is disclosed in U.S. Patent ApplicationPublication No. 2002 0123057 A1.

The invention thus provides a method of manufacturing an antibody orantigen binding fragment thereof comprising: (a) expressing one or morepolynucleotide molecule(s) encoding a VL and VH chain of an antibody ina cell; and (b) purifying the antibody from the cell, wherein theantibody specifically binds to a TCRα chain. In embodiments, theantibody or antigen binding fragment thereof selectively binds anepitope of TCRα chain comprising GSTLRG (SEQ ID NO:1).

VIII. METHOD OF SELECTING A TCR-CONTAINING CELL

In a further embodiment, there is provided a method for selecting a cellcomprising a T-cell receptor (TCR) comprising: (a) contacting the cellwith an antibody that binds to a TCR, wherein the antibody binds toT-cells having a plurality of T-cell epitope specificities; and (b)selecting a cell comprising the TCR based on binding of the antibody. Incertain aspects, the antibody: (i) competes for binding of TCR with the79A, 79A-23, 79A-15, 79A-11 or 79A-13 monoclonal antibody; (ii) binds toT-cells essentially independent of T-cell epitope specificity; (iii)agonizes TCR activity of T-cells; or (iv) stimulates T-cellproliferation.

In some aspects of the methods of selecting a TCR-containing cell, theantibody binds to a TCRα polypeptide. In other aspects, the antibodybinds to an epitope of the TCRα polypeptide comprising the sequenceGSTLRG (SEQ ID NO: 1). In some aspects, the cell is in vivo. In someaspects, the antibody comprises a reporter and the method comprisesselecting cells comprising a TCR by fluorescence-activated cell sorting(FACS). In some aspects, selecting cells comprises selecting usingparamagnetic beads. In certain aspects, the antibody is bound to asupport. For example, the support is a bead, a surface, a column or hostcell. In certain aspects, the method is further defined as a method forpurifying or enriching T-cells. In further aspects, contacting the cellwith an antibody comprises inducing activation or growth in the cellcomprising the TCR. In some aspects, purifying or enriching cellscomprising a TCR comprises stimulating proliferation of the cells.

IX. ARTIFICIAL ANTIGEN PRESENTING CELLS (aAPCs)

In some cases, artificial antigen presenting cells (“aAPCs”) alsoreferred to as activating and propagating cells (aAPCs) are useful inthe expansion, propagation and/or activation of T-cells or T-cellprogenitors and in preparing T-cell-based therapeutic compositions andcell therapy products. T-cell therapeutic compositions can include forexample, T cells that are genetically modified to include chimericantigen receptor (CAR), T cell receptors (TCR) or any chimericreceptors.

aAPCs for use according to the embodiments include but are not limitedto dendritic cells, macrophages, immortalized cells and artificialantigen presenting cells. In one aspect, the aAPCs may be transgenicK562 cells. In some aspects of the invention, the aAPCs are HLACnegative, and in some aspects of the invention, the aAPCs are HLACpositive. In certain aspects, aAPC are conjugated to an anti-TCR-bindingantibody of the embodiments. In further aspects, an aAPC comprises anexpression construct for surface expression of an anti-TCR-bindingantibody (e.g., a membrane-bound antibody). For general guidanceregarding the preparation and use of antigen-presenting systems, see,e.g., U.S. Pat. Nos. 6,225,042, 6,355,479, 6,362,001 and 6,790,662; U.S.Patent Application Publication Nos. 2009/0017000 and 2009/0004142; andInternational Publication No. W02007/103009, each of which isincorporated herein by reference.

The aAPCs may comprise additional molecules that activate orco-stimulate T-cells in some cases. The additional molecules may, insome cases, comprise membrane-bound Cy cytokines. In yet still furtheraspects, the aAPCs are inactivated or irradiated, or have been testedfor and confirmed to be free of infectious material. In still furtheraspects, culturing the cells in the presence of aAPCs comprisesculturing the cells in a medium comprising soluble cytokines, such asIL-15, IL-21 and/or IL-2. The cells may be cultured at a ratio of about10:1 to about 1:10; about 3:1 to about 1:5; about 1:1 to about 1:3(immune effector cells to aAPCs); or any range derivable therein. Forexample, the co-culture of T cells and aAPCs can be at a ratio of about1:1, about 1:2 or about 1:3. In some aspects, ex vivo culturing thegenetically modified T cells or T cells, is for no more than 14 days, nomore than 7 days or no more than 3 days. In other aspects, thegenetically modified T cells or T cells are cultured ex vivo for lessthan 21 days, such as for less than 18, 17, 16, 15, 14, 13, 12, 1 1, 10,9, 8, 7, 6, 5, 4, 3, 2 days, 1 day or less.

For example, the ex vivo culture (e.g., culture in the presence of AaPCsor aAPCs) can be performed for less than one population doubling of theT cells. In still further aspects, the T cells are not cultured ex vivoin the presence of AaPCs or aAPCs.

In one aspect, the aAPCs may express CD137L. In other aspects, the aAPCsmay further express CD19, CD64, CD86, or membrane bound IL-15. In someaspects, the membrane-bound IL-15 (mIL-15) comprises a fusion proteinbetween IL-15 and IL-15Ra. In a further aspect, the mIL-15 constructcomprises the IL-15 cDNA sequence (NM 000585.4) fused to the full lengthIL-15Ra (NM 002189.3) with a serine-glycine linker. An IgE signalpeptide (gb−AAB59424.1) can be used for the mIL15 fusion construct. Anexample of mIL-15 is described in WO/2014/186469, herein incorporated byreference.

In embodiments, a truncated CD8 transmembrane protein (tCD8-TM) ortruncated human Fc-tCD8-TM domain (with and without HA tag) or similarextracellular scaffolding molecule is fused to an anti-TCR antibody orfragment thereof and expressed on cells, such as derived from K562 alongwith co-stimulatory ligands C86, CD137L, or IL15 (including as amembrane-bound formulation) or any combination thereof. In aspects ofthe invention, SEQ ID NO:24 is an example of a human truncated CD8domain, SEQ ID NO:25 is an example of a human CD8a transmembrane domain,and SEQ ID NO:26 is an example of a hCD8a extracellular domain.

aAPCs may be used to expand and/or activate T cells generally or in aTCR epitope specific manner. During encounter with tumor antigen, thesignals delivered to T cells by antigen-presenting cells can affectT-cell programming and their subsequent therapeutic efficacy. This hasstimulated efforts to develop artificial antigen-presenting cells thatallow optimal control over the signals provided to T cells (Turtle etal., 2010). In addition to antibody, such as TCR-binding antibody, theAPC systems may also comprise at least one exogenous assisting molecule.Any suitable number and combination of assisting molecules may beemployed. The assisting molecule may be selected from assistingmolecules such as co-stimulatory molecules and adhesion molecules.Exemplary co-stimulatory molecules include C86, CD137L and mIL15 orOX40L (CD134L); along with CD70 and B7.1 (also called B7 or CD80), whichcan bind to CD28, 4-1BB or OX40 molecules on the surface of T cells,thereby affecting, e.g., T-cell expansion, Th1 differentiation,short-term T-cell survival, and cytokine secretion such as interleukin(IL)-2. Adhesion molecules may include carbohydrate-bindingglycoproteins such as selectins, transmembrane binding glycoproteinssuch as integrins, calcium-dependent proteins such as cadherins, andsingle-pass transmembrane immunoglobulin (Ig) superfamily proteins, suchas intercellular adhesion molecules (ICAMs), that promote, for example,cell-to-cell or cell-to-matrix contact. Exemplary adhesion moleculesinclude LFA-3 and ICAMs, such as ICAM-1. Techniques, methods, andreagents useful for selection, cloning, preparation, and expression ofexemplary assisting molecules, including co-stimulatory molecules andadhesion molecules, are exemplified in, e.g., U.S. Pat. Nos. 6,225,042,6,355,479, and 6,362,001, incorporated herein by reference.

In some cases, cells selected to become aAPCs, have deficiencies inintracellular antigen-processing, intracellular peptide trafficking,and/or intracellular MHC Class I or Class II molecule-peptide loading,or are poikilothermic (i.e., less sensitive to temperature challengethan mammalian cell lines), or possess both deficiencies andpoikilothermic properties. In some aspects, cells selected to becomeaAPCs also lack the ability to express at least one endogenouscounterpart (e.g., endogenous MHC Class I or Class II molecule and/orendogenous assisting molecules as described above) to the exogenous MHCClass I or Class II molecule and assisting molecule components that areintroduced into the cells. Furthermore, aAPCs may retain thedeficiencies and poikilothermic properties that were possessed by thecells prior to their modification to generate the aAPCs. Exemplary aAPCseither constitute or are derived from a transporter associated withantigen processing (TAP)-deficient cell line, such as an insect cellline. An exemplary poikilothermic insect cells line is a Drosophila cellline, such as a Schneider 2 cell line (e.g., Schneider, J.m 1972).Illustrative methods for the preparation, growth, and culture ofSchneider 2 cells, are provided in U.S. Pat. Nos. 6,225,042, 6,355,479,and 6,362,001, incorporated herein by reference.

aAPCs may be subjected to a freeze-thaw cycle. For example, aAPCs may befrozen by contacting a suitable receptacle containing the aAPCs with anappropriate amount of liquid nitrogen, solid carbon dioxide (dry ice),or similar low-temperature material, such that freezing occurs rapidly.The frozen aAPCs are then thawed, either by removal of the aAPCs fromthe low-temperature material and exposure to ambient room temperatureconditions, or by a facilitated thawing process in which a lukewarmwater bath or warm hand is employed to facilitate a shorter thawingtime. Additionally, aAPCs may be frozen and stored for an extendedperiod of time prior to thawing. Frozen aAPCs may also be thawed andthen lyophilized before further use. Preservatives that mightdetrimentally impact the freeze-thaw procedures, such as dimethylsulfoxide (DMSO), polyethylene glycols (PEGs), and other preservatives,may be advantageously absent from media containing aAPCs that undergothe freeze-thaw cycle, or are essentially removed, such as by transferof aAPCs to media that is essentially devoid of such preservatives.

In other embodiments, xenogenic nucleic acid and nucleic acid endogenousto the aAPCs may be inactivated by crosslinking, so that essentially nocell growth, replication or expression of nucleic acid occurs after theinactivation. For example, aAPCs may be inactivated at a pointsubsequent to the expression of exogenous MHC and assisting molecules,presentation of such molecules on the surface of the aAPCs, and loadingof presented MHC molecules with selected peptide or peptides.Accordingly, such inactivated and selected peptide loaded aAPCs, whilerendered essentially incapable of proliferating or replicating, mayretain selected peptide presentation function. The crosslinking can alsoresult in APCS that are essentially free of contaminatingmicroorganisms, such as bacteria and viruses, without substantiallydecreasing the antigen-presenting cell function of the aAPCs. Thuscrosslinking can be used to maintain the important APC functions ofaAPCs while helping to alleviate concerns about safety of a cell therapyproduct developed using the aAPCs. For methods related to crosslinkingand aAPCs, see for example, U.S. Patent Application Publication No.20090017000, which is incorporated herein by reference.

X. METHODS OF EXPANDING AND/OR ACTIVATING T CELLS

The invention provides methods of T-cell activation and ex vivo and invivo propagation to generate T cells. In embodiments concerning ex vivoactivation and propagation, the T-cells are clinical grade. Inembodiments, the methods activate T cells by αβ-TCR cross-linking usingthe antibodies or antigen binding fragments of the invention. Inembodiments, the T-cell is a peripheral blood mononuclear cell (PBMC).In further embodiments, T cells can include for example, T cells thatare genetically modified to include chimeric antigen receptor (CAR), Tcell receptors (TCR) or any chimeric receptors. In some aspects, the Tcells may also be modified with cytokines that stimulate proliferationand/or survival of T cells. For example, the T cells can comprisemembrane-bound versions of IL-7, IL-15 or IL-21. In some aspects, thecytokine is tethered to the membrane by fusion of the cytokine codingsequence with the receptor for the cytokine. For example, a cell cancomprise a vector for expression of a IL-15-IL-15Ra fusion protein, seee.g. WO 2014186469, herein incorporated by reference.

In further aspects, the T cells are further modified with a transposasethat facilitates integration of a CAR or TCR coding sequence into thegenome of the transfected cell. In some aspects, the transposase isprovided as DNA expression vector. However, in preferred aspects, thetransposase is provided as an expressible RNA or a protein such thatlong-term expression of the transposase does not occur in the transgeniccells. For example, in some aspects, the transposase is provided as anmRNA (e.g., an mRNA comprising a cap and poly-A tail). Any transposasesystem may be used in accordance with the embodiments. However, in someaspects, the transposase is salmonid-type Tel-like transposase (SB). Forexample, the transposase can be the so called “Sleeping beauty”transposase, see e.g., U.S. Pat. No. 6,489,458, incorporated herein byreference. In certain aspects, the transposase is an engineered enzymewith increased enzymatic activity. Some specific examples oftransposases include, without limitation, SB 10, SB 11, SB 100×transposase (see, e.g., Mates et ah, 2009, incorporated herein byreference) or SB110× tranposase. For example, a method can involveelectroporation of cells with a mRNA encoding a SB 10, SB 11, SB100× orSB110× transposase.

Activating T cells, or T cell activation, includes numeric expansion ofT cells (i.e., increasing the number of T cells) (also referred to as Tcell expansion) as well as stimulating T cells to produce cytokine andto kill specific cells (e.g., tumor antigen presenting cells).

The methods of expanding and/or activating T-cells of the invention canbe used in a clinical setting to expand and/or activate the T cellpopulation of a diseased subject, i.e., a subject in need thereof, or toexpand and/or activate the T cell population of a donor, i.e., T cellsderived from a source other than the diseased subject.

The invention additionally provides methods for expanding and/oractivating T-cells comprising contacting the T-cells with artificialantigen presenting cells (aAPCs) in the presence of an antibody orantigen binding fragment thereof that binds to an epitope of a T-cellReceptor (TCR), wherein said epitope is a polypeptide comprisingsequence GSTLRG (SEQ ID NO:1). In embodiments, the aAPCs are conjugatedto an antibody or antigen-binding fragment thereof that binds to the TCRepitope. In embodiments, the aAPCs comprise an expression construct forexpressing an antibody or antigen binding fragment thereof that binds tosaid epitope of a TCR. In embodiments, the antibody or antigen bindingfragment thereof engages αβ-TCR in a conformation specific manner toelicit polyclonal T cell response through recognition via plurality ofT-cell epitope specificities.

In the methods of expanding and/or activating T cells, as disclosedherein, in embodiments, the antibody or antigen-binding fragment is ananti-TCR antibody as disclosed herein. For example, the antibody orantigen-binding fragment thereof comprises HCDR1 at least 95% or 100%identical to SEQ ID NO:2; HCDR2 at least 95% identical to SEQ ID NO:3;HCDR3 identical to CAYL (SEQ ID NO:22). In an additional embodiment, theantibody or antigen-binding fragment thereof comprises LCDR1 at least95% or 100% identical to SEQ ID NO:9; LCDR2 at least 95% or 100%identical to SEQ ID NO:5; LCDR3 at least 95% identical to SEQ ID NO:10.Further, the antibody or antigen-binding fragment thereof comprises aHCDR1 at least 95% or 100% identical to SEQ ID NO:2; HCDR2 at least 95%or 100% identical to SEQ ID NO:3; HCDR3 identical to CAYW (SEQ IDNO:23).

In the methods of expanding and/or activating T cells, as disclosedherein, in embodiments, the antibody or antigen-binding fragment is ananti-TCR antibody as disclosed herein. For example, the antibody orantigen-binding fragment thereof comprises LCDR1 at least 95% or 100%identical to SEQ ID NO:9; LCDR2 at least 95% or 100% identical to SEQ IDNO:5; LCDR3 at least 95% or 100% identical to SEQ ID NO:10. Inembodiments, the antibody or antigen-binding fragment thereof comprisesLCDR1 at least 95% or 100% identical to SEQ ID NO:4; LCDR2 at least 95%or 100% identical to SEQ ID NO:5; LCDR3 at least 95% or 100% identicalto SEQ ID NO:10. In additional embodiments, the antibody orantigen-binding fragment thereof comprises HCDR1 at least 95% or 100%identical to SEQ ID NO:15; HCDR2 at least 95% or 100% identical to SEQID NO:16; HCDR3 identical to CRYW (SEQ ID NO:17). In furtherembodiments, the antibody or antigen-binding fragment thereof comprisesLCDR1 at least 95% or 100% identical to SEQ ID NO:18; LCDR2 at least 95%or 100% identical to SEQ ID NO:5; LCDR3 at least 95% or 100% identicalto SEQ ID NO:6.

In the methods of expanding and/or activating T cells, as disclosedherein, in embodiments, the antibody or antigen-binding fragment is ananti-TCR antibody as disclosed herein. For example, the antibody orantigen binding fragment comprises a VH and VL domain separated by alinker. In embodiments, the antibody or antigen binding fragment furthercomprises a transmembrane domain (e.g., hCDalpha (SEQ ID NO:25)). In themethods of expanding and/or activating T cells, as disclosed herein, inembodiments, the antibody or antigen-binding fragment is an anti-TCRantibody as disclosed herein. For example, the VH is a polypeptide atleast 90% identical to a polypeptide selected from the group consistingof a SEQ ID NO:11, SEQ ID NO:13 and SEQ ID NO:19, and the VL is apolypeptide at least 90% identical to a polypeptide selected from thegroup consisting of SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:20. Inembodiments, the VH is a polypeptide at least 90% identical to SEQ IDNO:11 and said VL is a polypeptide at least 90% identical to SEQ IDNO:12. In further embodiments, the VH is a polypeptide at least 90%identical to SEQ ID NO:13 and said VL is a polypeptide at least 90%identical to SEQ ID NO:12. In additional embodiments, the VH is apolypeptide at least 90% identical to SEQ ID NO:11 and said VL is apolypeptide at least 90% identical to SEQ ID NO:14. In a furtherembodiment, the VH is a polypeptide at least 90% identical to SEQ IDNO:19 and said VL is a polypeptide at least 90% identical to SEQ IDNO:20.

In certain aspects of the invention, stimulation of primary T cells isachieved by using anti-TCR soluble antibodies or antigen bindingfragments of the invention in culture. In other aspects, sustainedactivation is done by engaging the αβ-TCR of T cells via the solubleanti-TCR antibodies or antigen binding fragments of the invention in thepresence of T cell growth promoting cytokines such as IL-2, IL-7, IL-15or IL-21. In other aspects, T cells are numerically expanded by constantengagement of the TCR ligand through a support (such as magneticbeads/polystyrene surface or through cells like irradiated K562 feedercells). Culture conditions and mode of ligand presentation will vary toimpact activation and stimulation leading to T cell numeric expansion.

XI. TREATMENT METHODS USING THERAPEUTIC ANTI-TCR ANTIBODIES

The invention also provides methods of treating an autoimmune disease ora T cell leukemia or T cell lymphoma, or preventing/controllingrejection of an allogeneic graft (bone marrow or solid organ) in ananimal in need of treatment, comprising administering to the animal theanti-TCR antibody and/or antigen binding fragment of the invention as asoluble protein, a portion of the antibody, or as a host cell comprisinga chimeric antigen receptor (CAR) targeting TCRα polypeptide comprisingsequence GSTLRG (SEQ ID NO:1). In embodiments, the host cell is an NK orgamma delta T cell or T cell that has been engineered to preventexpression of endogenous alpha beta TCR.

A CAR is typically composed of an extracellular antibody-derivedsingle-chain variable domain (scFv) for antigen recognition and isjoined by a flexible linker connected to a transmembrane domain and anintracellular signaling domain(s) that includes CD3ζ for T-cellactivation. Normally when T cells are activated in vivo they receive aprimary antigen induced TCR signal with secondary costimulatorysignaling from CD28 that induces the production of cytokines (i.e., IL-2and IL-21), which then feed back into the signaling loop in anautocrine/paracrine fashion. With this in mind, CARs can include CD28cytoplasmic signaling domain or other costimulatory molecule signalingdomains such as 4-1BB signaling domain. Chimeric CD28 co-stimulationimproves T-cell persistence by up-regulation of anti-apoptotic moleculesand production of IL-2, as well as expanding T cells derived fromperipheral blood mononuclear cells (PBMC).

In one embodiment, CARs are fusions of single-chain variable fragments(scFv) derived from monoclonal antibodies fused to transmembrane domainand CD3-zeta endodomain. Such molecules result in the transmission of azeta signal in response to recognition by the scFv of its target.

In an embodiment, a CAR may have an ectodomain (extracellular), atransmembrane domain and an endodomain (intracellular). In oneembodiment of the CAR ectodomain, a signal peptide directs the nascentprotein into the endoplasmic reticulum. This is if the receptor is to beglycosylated and anchored in the cell membrane for example. Anyeukaryotic signal peptide sequence is envisaged to be functional.Generally, the signal peptide natively attached to the amino-terminalmost component is used (e.g. in a scFv with orientation lightchain-linker-heavy chain, the native signal of the light-chain is used).Exemplary signal peptides that can be used include signal peptides fromimmunoglobulin, CD3, CD3ζ, CD8alpha (CD8a) and CD28.

The antigen recognition domain may be a scFv. There may however bealternatives. An antigen recognition domain from native T-cell receptor(TCR) alpha and beta single chains are envisaged, as they have simpleectodomains (e.g. CD4 ectodomain to recognize HIV infected cells) and aswell as other recognition components such as a linked e.g., cytokine(which leads to recognition of cells bearing the cytokine receptor).Almost anything that binds a given target with high affinity can be usedas an antigen recognition region. In further aspects, the scFvs can bederived from SEQ ID NOs: 7, 11, 13, 19, 20, 8, 12, 14, 19 and/or 20. Infurther aspects, the sFcvs comprise SEQ ID NOs: 7 and 8; SEQ ID NOs: 11and 12; SEQ ID NOs:13 and 14; or SEQ ID NOs:19 and 20, in each caseoptionally separated by a linker. In yet further aspects, the scFvsinclude SEQ ID NOs: 2, 3, 21, 4, 5, and/or 6; SEQ ID NOs: 2, 3, 22, 9, 5and/or 10; SEQ ID NOs: 2, 3, 23, 9, 5 and/or 10; SEQ ID NOs: 2, 3, 22,4, 5 and/or 10; or SEQ ID NOs: 15, 16, 17, 18, 5 and/or 6.

In general, CARs exist in a dimerized form and are expressed as a fusionprotein that links the extracellular scFv (VH linked to VL) region, astalk domain, a transmembrane domain, and intracellular signalingmotifs. The endodomain of the first generation CAR induces T cellactivation solely through CD3-ζ signaling. The second generation CARprovides activation signaling through CD3-ζ and CD28, or otherendodomains such as 4-1BB or OX40. The 3rd generation CAR activates Tcells via a CD3-ζ-containing combination of three signaling motifs suchas CD28, 4-1BB, or OX40.

In embodiments, between the extracellular domain and the transmembranedomain of the CAR, there is incorporated a stalk domain. As used herein,the term “stalk domain” generally means any oligonucleotide- orpolypeptide that functions to link the transmembrane domain to, eitherthe scFv or, the cytoplasmic domain in the polypeptide chain. A stalkdomain can include a flexible hinge such as a Fc hinge and optionallyone or two constant domains of Fc.

In aspects of the invention, the sequence of the anti-TCR antibody orantigen binding fragment thereof is used to redirect the specificity orbinding of an immune cell. For example, the antigen-binding sequence ofthe anti-TCR antibody or antigen binding fragment of the invention isused to create a membrane-bound protein which when expressed on animmune cell can be used to redirect the specificity or binding of saidimmune cell.

In additional embodiments, the invention provides methods of treatingdisease, such as cancer or infection by a pathogen in an animal in needof treatment, comprising administering to the animal a multi-specific Tcell engager comprising (a) an antibody or antigen binding fragmentthereof targeting a TCRα polypeptide comprising sequence GSTLRG (SEQ IDNO:1), and (b) at least one antibody or antigen binding fragment thereoftargeting a tumor associated antigen. An exemplary antibody or antigenbinding fragment thereof targeting a TCRα polypeptide comprisingsequence GSTLRG (SEQ ID NO:1) is an scFv. An exemplary antibody orantigen binding fragment thereof targeting a tumor associated antigen isan scFv. An exemplary multi-specific T cell engager is a bispecific Tcell engager comprising an scFv targeting the TCRα polypeptide and anscFv targeting a tumor associated antigen. A trispecific T cell engager,for example, comprises an scFv targeting the TCRα polypeptide and afirst and second scFv targeting tumor associated antigens. In furtheraspects, the scFv targeting the TCRα polypeptide in this embodimentcomprises SEQ ID NOs: 7, 8, 11, 12, 13, 14, 19, and/or 20. In furtheraspects, the scFv comprises SEQ ID NOs: 7 and 8; SEQ ID NOs: 11 and 12;SEQ ID NOs: 13 and 14; or SEQ ID NOs: 19 and 20, in each case thesequences are optionally separated by a linker. In yet further aspects,the scFvs comprise SEQ ID NOs: 2, 3, 21, 4, 5, and/or 6; SEQ ID NOs: 2,3, 22, 9, 5 and/or 10; SEQ ID NOs: 2, 3, 23, 9, 5 and/or 10; SEQ ID NOs:2, 3, 22, 4, 5 and/or 10; or SEQ ID NOs: 15, 16, 17, 18, 5 and/or 6.

In other embodiments, the exemplary antibody or antigen binding fragmentthereof targeting a TCRα polypeptide comprising sequence GSTLRG (SEQ IDNO:1) may be combined with other scFvs or proteins that bind tumorantigen to create a soluble recombinant “binder”. Upon engaging tumor,the binder can activate T cells by cross-linking TCR to activate T cellsfor effector functions such as proliferation, cytokine production andkilling.

In the treatment methods of the invention, in certain aspects, theanimal in need of treatment is a human.

In certain embodiments, the multi-specific T-cell engager comprises anantibody or antigen binding fragment thereof targeting a tumorassociated antigen or a pathogen-specific antigen binding domainincluding carbohydrate antigen recognized by pattern-recognitionreceptors, such as Dectin-1. A tumor associated antigen may be of anykind so long as it is expressed on the cell surface of tumor cells.Exemplary embodiments of tumor associated antigens include CD19, CD20,carcinoembryonic antigen, alphafetoprotein, CA-125, MUC-1, CD56, EGFR,c-Met, AKT, Her2, Her3, epithelial tumor antigen, melanoma-associatedantigen, mutated p53, mutated ras, and so forth.

In certain embodiments intracellular tumor associated antigens may betargeted, such as HA-1, survivin, WT1, and p53. This can be achieved bya scFv expressed on a universal T cell that recognizes the processedpeptide described from the intracellular tumor associated antigen in thecontext of HLA. In addition, the universal T cell may be geneticallymodified to express a T-cell receptor pairing that recognizes theintracellular processed tumor associated antigen in the context of HLA.In certain embodiments, the T cell can be co-expressed with amembrane-bound cytokine to improve persistence when there is a lowamount of tumor-associated antigen. For example, CAR can be co-expressedwith membrane-bound IL-15.

In embodiments, the anti-TCR antibodies and antigen binding fragments ofthe invention suppress the immune system in a subject in need thereof invivo, for example by over-activating T cells, leading to the loss of Tcells and immune-suppression. For instance, organ, bone marrow and stemcell transplantation can improve quality of life and prolong survival.However, rejection of transplanted tissues requires suppression of theimmune system by various means. The invention therefore provides methodsof suppressing the immune system comprising administering to a patientin need thereof an anti-TCR antibody or antigen binding fragmentthereof. In embodiments, the anti-TCR antibody or antigen bindingfragment thereof of the invention is administered to a patient in needthereof via intravenous (IV) injection once per day, twice a day orthree times a day for 2 to 21 days at a dosage of 1 to 20 mg per day. Inembodiments, anti-TCR antibody or antigen binding fragment thereof ofthe invention is administered to a patient in need thereof via IVinjection once per day for 5 to 15 days at a dosage of 5 to 10 mg/day.In embodiments, anti-TCR antibody or antigen binding fragment thereof ofthe invention is administered to a patient in need thereof via IVinjection once per day for 10 to 14 days at a dosage of 5 mg/day.

In additional embodiments, the anti-TCR antibodies and antigen bindingfragments of the invention are administered to a subject in need thereofin a therapeutic method to activate T cells in vivo, and/or for in vivoeffector functions, such as antibody-dependent cellular cytotoxicity(ADCC) and complement-dependent cytotoxicity (CDC). In embodiments, theanti-TCR antibody or antigen binding fragment thereof of the inventionis administered to a patient in need thereof via intravenous (IV)injection once per day, twice a day or three times a day for 2 to 21days at a dosage of 1 to 20 mg per day. In embodiments, anti-TCRantibody or antigen binding fragment thereof of the invention isadministered to a patient in need thereof via IV injection once per dayfor 5 to 15 days at a dosage of 5 to 10 mg/day. In embodiments, anti-TCRantibody or antigen binding fragment thereof of the invention isadministered to a patient in need thereof via IV injection once per dayfor 10 to 14 days at a dosage of 5 mg/day.

XII. PHARMACEUTICAL COMPOSITIONS AND ADMINISTRATION METHODS

Methods of preparing and administering the anti-TCR binding molecule,e.g., antibodies, or antigen-binding fragments, variants, or derivativesthereof, of the invention to a subject in need thereof are well known toor are readily determined by those skilled in the art. The route ofadministration of the anti-TCR binding molecule, e.g, antibody, orantigen-binding fragment, variant, or derivative thereof, may be, forexample, oral, parenteral, by inhalation or topical. The term parenteralas used herein includes, e.g., intravenous, intraarterial,intraperitoneal, intramuscular, subcutaneous, rectal, or vaginaladministration. While all these forms of administration are clearlycontemplated as being within the scope of the invention, an example of aform for administration would be a solution for injection, in particularfor intravenous or intraarterial injection or drip. Usually, a suitablepharmaceutical composition for injection may comprise a buffer (e.g.acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate),optionally a stabilizer agent (e.g. human albumin), etc. However, inother methods compatible with the teachings herein, anti-TCR bindingmolecules, e.g., antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can be delivered directly to thesite of the adverse cellular population thereby increasing the exposureof the diseased tissue to the therapeutic agent.

As discussed herein, anti-TCR binding molecules, e.g., antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention may be administered in a pharmaceutically effective amount forthe in vivo treatment of TCR-expressing cell-mediated diseases such ascertain types of cancers, autoimmune diseases, inflammatory diseasesincluding central nervous system (CNS) and peripheral nervous system(PNS) inflammatory diseases, and invasive angiogenesis. In this regard,it will be appreciated that the disclosed binding molecules of theinvention will be formulated so as to facilitate administration andpromote stability of the active agent. Preferably, pharmaceuticalcompositions in accordance with the present invention comprise apharmaceutically acceptable, non-toxic, sterile carrier such asphysiological saline, non-toxic buffers, preservatives and the like. Forthe purposes of the instant application, a pharmaceutically effectiveamount of an anti-TCR binding molecule, e.g., an antibody, orantigen-binding fragment, variant, or derivative thereof conjugated orunconjugated, shall be held to mean an amount sufficient to achieveeffective binding to a target and to achieve a benefit, e.g., toameliorate symptoms of a disease or disorder or to detect a substance ora cell.

The pharmaceutical compositions used in this invention comprisepharmaceutically acceptable carriers, including, e.g., ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances such as phosphates, glycine, sorbicacid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium tri silicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol, andwool fat.

Preparations for parenteral administration includes sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include, e.g., water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. In the subject invention, pharmaceutically acceptable carriersinclude, but are not limited to, 0.01-0.1 M and preferably 0.05 Mphosphate buffer or 0.8% saline. Other common parenteral vehiclesinclude sodium phosphate solutions, Ringer's dextrose, dextrose andsodium chloride, lactated Ringer's, or fixed oils. Intravenous vehiclesinclude fluid and nutrient replenishers, electrolyte replenishers, suchas those based on Ringer's dextrose, and the like. Preservatives andother additives may also be present such as, for example,antimicrobials, antioxidants, chelating agents, and inert gases and thelike.

More particularly, pharmaceutical compositions suitable for injectableuse include sterile aqueous solutions (where water soluble) ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions. In such cases, thecomposition must be sterile and should be fluid to the extent that easysyringability exists. It should be stable under the conditions ofmanufacture and storage and will preferably be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Suitableformulations for use in the therapeutic methods disclosed herein aredescribed in Remington's Pharmaceutical Sciences (Mack Publishing Co.)16th ed. (1980).

Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

In any case, sterile injectable solutions can be prepared byincorporating an active compound (e.g., an anti-TCR antibody, orantigen-binding fragment, variant, or derivative thereof, by itself orin combination with other active agents) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedherein, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle, which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-drying,which yields a powder of an active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The preparations for injections are processed, filled into containerssuch as ampoules, bags, bottles, syringes or vials, and sealed underaseptic conditions according to methods known in the art. Further, thepreparations may be packaged and sold in the form of a kit such as thosedescribed in U.S. Publ. No. 2002/0102208. Such articles of manufacturewill preferably have labels or package inserts indicating that theassociated compositions are useful for treating a subject sufferingfrom, or predisposed to a disease or disorder.

Parenteral formulations may be a single bolus dose, an infusion or aloading bolus dose followed with a maintenance dose. These compositionsmay be administered at specific fixed or variable intervals, e.g., oncea day, or on an “as needed” basis.

Certain pharmaceutical compositions used in this invention may be orallyadministered in an acceptable dosage form including, e.g., capsules,tablets, aqueous suspensions or solutions. Certain pharmaceuticalcompositions also may be administered by nasal aerosol or inhalation.Such compositions may be prepared as solutions in saline, employingbenzyl alcohol or other suitable preservatives, absorption promoters toenhance bioavailability, and/or other conventional solubilizing ordispersing agents.

The amount of an anti-TCR binding molecule, e.g., antibody, or fragment,variant, or derivative thereof, that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. The compositionmay be administered as a single dose, multiple doses or over anestablished period of time in an infusion. Dosage regimens also may beadjusted to provide the optimum desired response (a therapeutic orprophylactic response).

In keeping with the scope of the present disclosure, anti-TCRantibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention may be administered to a human or other animalin accordance with the aforementioned methods of treatment in an amountsufficient to produce a therapeutic effect. The anti-TCR antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention can be administered to such human or other animal in aconventional dosage form prepared by combining the antibody of theinvention with a conventional pharmaceutically acceptable carrier ordiluent according to known techniques. It will be recognized by one ofskill in the art that the form and character of the pharmaceuticallyacceptable carrier or diluent is dictated by the amount of activeingredient with which it is to be combined, the route of administrationand other well-known variables. Those skilled in the art will furtherappreciate that a cocktail comprising one or more species of anti-TCRbinding molecules, e.g., antibodies, or antigen-binding fragments,variants, or derivatives thereof, of the invention may prove to beparticularly effective.

By “therapeutically effective dose or amount” or “effective amount” isintended an amount of anti-TCR binding molecule, e.g., antibody orantigen binding fragment thereof, that when administered brings about apositive therapeutic response with respect to treatment of a patientwith a disease to be treated.

Therapeutically effective doses of the compositions of the presentinvention, for treatment of TCR-expressing cell-mediated diseases suchas certain types of cancers, e.g., head and neck, prostate, colon,breast, and lung cancers; autoimmune diseases, e.g., arthritis, multiplesclerosis, inflammatory diseases including central nervous system (CNS)and peripheral nervous system (PNS) inflammatory diseases; and invasiveangiogenesis, vary depending upon many different factors, includingmeans of administration, target site, physiological state of thepatient, whether the patient is human or an animal, other medicationsadministered, and whether treatment is prophylactic or therapeutic.Usually, the patient is a human, but non-human mammals includingtransgenic mammals can also be treated. Treatment dosages may betitrated using routine methods known to those of skill in the art tooptimize safety and efficacy.

The amount of at least one anti-TCR binding molecule, e.g., antibody orbinding fragment thereof, to be administered is readily determined byone of ordinary skill in the art without undue experimentation given thedisclosure of the present invention. Factors influencing the mode ofadministration and the respective amount of at least one anti-TCRbinding molecule, e.g., antibody, antigen-binding fragment, variant orderivative thereof include, but are not limited to, the severity of thedisease, the history of the disease, and the age, height, weight,health, and physical condition of the individual undergoing therapy.Similarly, the amount of anti-TCR binding molecule, e.g., antibody, orfragment, variant, or derivative thereof, to be administered will bedependent upon the mode of administration and whether the subject willundergo a single dose or multiple doses of this agent.

The present invention also provides for the use of an anti-TCR bindingmolecule, e.g., an antibody or antigen-binding fragment, variant, orderivative thereof, in the manufacture of a medicament for treating anautoimmune disease and/or inflammatory disease, including, e.g.,arthritis, multiple sclerosis, CNS and PNS inflammatory diseases, or acancer.

The invention also provides for the use of an anti-TCR binding molecule,e.g., antibody of the invention, or antigen-binding fragment, variant,or derivative thereof, in the manufacture of a medicament for treating asubject for treating an autoimmune disease and/or inflammatory disease,or for treating a cancer, wherein the medicament is used in a subjectthat has been pretreated with at least one other therapy. By“pretreated” or “pretreatment” is intended the subject has received oneor more other therapies (e.g., been treated with at least one othercancer therapy) prior to receiving the medicament comprising theanti-TCR binding molecule, e.g., antibody or antigen-binding fragment,variant, or derivative thereof, “Pretreated” or “pretreatment” includessubjects that have been treated with at least one other therapy within 2years, within 18 months, within 1 year, within 6 months, within 2months, within 6 weeks, within 1 month, within 4 weeks, within 3 weeks,within 2 weeks, within 1 week, within 6 days, within 5 days, within 4days, within 3 days, within 2 days, or even within 1 day prior toinitiation of treatment with the medicament comprising the anti-TCRbinding molecule, for example, the monoclonal antibody disclosed herein,or antigen-binding fragment, variant, or derivative thereof. It is notnecessary that the subject was a responder to pretreatment with theprior therapy or therapies. Thus, the subject that receives themedicament comprising the anti-TCR binding molecule, e.g., an antibodyor antigen-binding fragment, variant, or derivative thereof could haveresponded, or could have failed to respond (e.g., the cancer wasrefractory), to pretreatment with the prior therapy, or to one or moreof the prior therapies where pretreatment comprised multiple therapies.Examples of other cancer therapies for which a subject can have receivedpretreatment prior to receiving the medicament comprising the anti-TCRbinding molecule, e.g., antibody or antigen-binding fragment, variant,or derivative thereof include, but are not limited to, surgery;radiation therapy; chemotherapy, optionally in combination withautologous bone marrow transplant, where suitable chemotherapeuticagents include, but are not limited to, those listed herein above; otheranti-cancer monoclonal antibody therapy; small molecule-based cancertherapy, including, but not limited to, the small molecules listedherein above; vaccine/immunotherapy-based cancer therapies; steroidtherapy; other cancer therapy; or any combination thereof. The practiceof the present invention will employ, unless otherwise indicated,conventional techniques of cell biology, cell culture, molecularbiology, transgenic biology, microbiology, recombinant DNA, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature. See, for example, Sambrook et al.,ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold SpringHarbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning:A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D. N. Glovered., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984)Oligonucleotide Synthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hamesand Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins,eds. (1984) Transcription And Translation; Freshney (1987) Culture OfAnimal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRLPress) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; thetreatise, Methods ID Enzymology (Academic Press, Inc., N.Y.); Miller andCalos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (ColdSpring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols.154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods IDCell And Molecular Biology (Academic Press, London); Weir and Blackwell,eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV;Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986); and in Ausubel et al. (1989) CurrentProtocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

General principles of antibody engineering are set forth in Borrebaeck,ed. (1995) Antibody Engineering (2nd ed.; Oxford Univ. Press). Generalprinciples of protein engineering are set forth in Rickwood et al., eds.(1995) Protein Engineering, A Practical Approach (IRL Press at OxfordUniv. Press, Oxford, Eng.). General principles of antibodies andantibody-hapten binding are set forth in: Nisonoff (1984) Molecularimmunology (2nd ed.; Sinauer Associates, Sunderland, Mass.); and Steward(1984) Antibodies, Their Structure and Function (Chapman and Hall, NewYork, N.Y.). Additionally, standard methods in immunology known in theart and not specifically described are generally followed as in CurrentProtocols in Immunology, John Wiley & Sons, New York; Stites et al.,eds. (1994) Basic and Clinical Immunology (8th ed; Appleton & Lange,Norwalk, Conn.) and Mishell and Shiigi (eds) (1980) Selected Methods inCellular Immunology (WIT. Freeman and Co., NY).

Standard reference works setting forth general principles of immunologyinclude Current Protocols in Immunology, John Wiley & Sons, New York;Klein (1982) J., Immunology: The Science of Self-Nonself Discrimination(John Wiley & Sons, NY); Kennett et al., eds. (1980) MonoclonalAntibodies, Hybridoma: A New Dimension in Biological Analyses (PlenumPress, NY); Campbell (1984) “Monoclonal Antibody Technology” inLaboratory Techniques in Biochemistry and Molecular Biology, ed. Burdenet al., (Elsevere, Amsterdam); Goldsby et al., eds. (2000) KubyImmunnology (4th ed.; H. Freemand & Co.); Roitt et al, (2001) Immunology(6th ed.; London: Mosby); Abbas et al, (2005) Cellular and MolecularImmunology (5th ed.; Elsevier Health Sciences Division); Kontermann andDubel (2001) Antibody Engineering (Springer Verlan); Sambrook andRussell (2001) Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Press); Lewin (2003) Genes VIII (Prentice Hall 2003); Harlow andLane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Press);Dieffenbach and Dveksler (2003) PCR Primer (Cold Spring Harbor Press).

All of the references cited above, as well as all references citedherein, are incorporated herein by reference in their entireties.

III. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1—Ex-Vivo Generation of T Cells and Analysis

Hybridoma—CD1D knockout mice were immunized with mouse L-fibroblastcells expressing fusion protein encoding TCR invariant chain(ICVVSDRGSTLGRL) fused to mCD8a. Hybridoma clones were generated viastandard cell fusion method using PEG-1500. Antibody clones specific toinvariant chain epitope were selected after screening on L-cellsexpressing the immunogen. Positive binders were verified with syntheticpeptides representing the immunogen by indirect ELISA coated with 100 ngof free peptides (FIG. 1A). A best binder 79A with highest OD450 waschosen for further analysis.

Antibody Generation and characterization—A monoclonal antibody (Clone79A) with narrow linear epitope specificity towards TCRVa24-Ja1 8junction (Amino acid Sequence: GSTLGR (SEQ ID NO:1)) but no apparentconformation specificity towards TCRα-CDR3 α-chain was chosen forantibody modeling and affinity improvement. Specificity of antibodyclone 79A was checked by ELISA on linear peptides, confirmed on anala-peptide library and also by flow cytometry analysis (FIGS. 1B-1D).

Binding Assay—Binding Assay was Performed on Solid Phase ELISA coatedwith linear peptides sequence spanning Va24-Ja18 regions (FIG. 1B). Flowcytometry was carried out on PBMC derived live CD3+ T cells using 79A ormutant antibody as primary antibody and matched fluorescent conjugatedsecondary antibody (either anti-mouse Fab PE or anti-human gamma Fc PEgoat Fab polyclonal) for detection (FIG. 1E). Western blot was done ondenatured proteins isolated from whole cell lysis (either PBMC, T Cells)resolved on SDS-PAGE.

V-gene amplification and scFv generation—Clone 79A specific to linearepitope GSTLGR (SEQ ID NO:1) was chosen for scFv generation. AntibodyV-genes were amplified from 79A cDNA by PCR involving standard mix ofdegenerate primers of murine VH FR1 for the 5′ primers and Ig constantregions for 3′ primers (FIG. 2) (Wang, Z., Raifu, M., Howard, M., Smith,L., Hansen, D., Goldsby, R. and Ratner, D. (2000) Universal PCRamplification of mouse immunoglobulin gene variable regions: the designof degenerate primers and an assessment of the effect of DNA polymerase3′ to 5′ exonuclease activity. Journal of immunological methods, 233,167-177). Correct size of VH/VL amplicons were verified by agarose gelelectrophoresis. PCR amplified V-gene products were excised from agarosegel, purified by gel purification kit (Qiagen) and the PCR product wascloned into TOPO vector as per manufacturer's instructions. Sequencingfor antibody genes was done by Sanger sequencing methods using eitherT3/T7 or M13F, M13R primers (MD Anderson DNA Sequencing Core). At least4 clones were used to obtain the consensus sequence of VH and VL on aNCBI BLAST. Antibody CDRs were identified by using consensus VH or VLsequences analyzed with help of IMGT/V-Quest software (available on theWorld Wide Web at imgt.org/IMGT_vquest/vquest). (Giudicelli, V., Chaume,D. and Lefranc, M. P. (2004) IMGT/V-QUEST, an integrated softwareprogram for immunoglobulin and T cell receptor V-J and V-D-Jrearrangement analysis. Nucleic acids research, 32, W435-440) CDRpositions were further confirmed by another software named V-Base(available on the World Wide Web at vbase2.org).

Molecular modeling and docking—Antibody homology modeling and dockingwas used to study the energetic contribution of residues in antibody(79A) CDRs with linear epitope specificity towards TCR-alpha chaininvariant region. 79A scFv sequences were subjected to ABR analysis byParatome server (FIG. 3A) and confirmed for correct positioning of CDRresidues in the VH and VL region. Antibody homology models weredeveloped by WAM server (Whitelegg, N. R. and Rees, A. R. (2000) WAM: animproved algorithm for modelling antibodies on the WEB. Proteinengineering, 13, 819-824) and SWISS-Models (FIG. 3B). SWISS-MODEL(Biasini, M., Bienert, S., Waterhouse, A., Arnold, K., Studer, G.,Schmidt, T., Kiefer, F., Cassarino, T. G., Bertoni, M., Bordoli, L. etal. (2014) SWISS-MODEL: modelling protein tertiary and quaternarystructure using evolutionary information. Nucleic acids research, 42,W252-258) was adapted to build V_(H) domain as A79 contains a very shortCDR3 in its heavy chain. Quality of the final antibody model structurewas checked with PROCHECK. 79A and TCR-models (PDB source) were used onZDOCK server to make a docking model (FIG. 3A, 3B, 3C). In the modelpercent amino acid residues located in the most favored or generousregions of the Ramachandran plot was 99.4%. Docking model of A79 and TCR[Protein Data Bank (PDB) ID code 1KGC4)] was built on ZDOCK server (FIG.3D). Pierce, B. G., Wiehe, K., Hwang, H., Kim, B. H., Vreven, T. andWeng, Z. (2014) ZDOCK server: interactive docking prediction ofprotein-protein complexes and symmetric multimers. Bioinformatics(Oxford, England), 30, 1771-1773. For the analysis of docked model, FoldX empirical force field was applied. Complex “Ala scan” function of FoldX was used to identify amino acid residues that could potentially bemutated to increase binding affinity. Mutated A79 models were generatedusing the “Build Model” function. Change in interaction energy (ΔΔG) wascalculated as *(ΔG=ΔGMU−ΔGWT) and then used to define energycontribution of relevant AA (in the CDRs of antibody binding region)towards cognate receptor. The total energy was defined as the bindingenergy of mutant minus the binding energy of wild type.

Complex-ALA-Scan function of FoldX software was used to identifypotential amino acid (AA) residues for mutation so that affinityenhancement in 79A CDRs towards TCR-alpha chain could be possible (FIGS.4A, 4C).

Molecular construct and antibody expression cassette—Schematic ofrecombinant antibody expression vector (encoding A79 mutant scFv fusedto truncated IgG₁ Fc), i.e., vector diagram is provided in FIGS. 5A and5B. In brief, antibody gene expression cassette consists of VH5 signalpeptide joined in frame to variable heavy chain (VH-mutant) and variablelight chain (VL-mutant). VH and VL are linked by a modified glycineserine G3S sequence to generate A79-scFv. Four such mutants were made(S11, S13, S15 and S23). Out of four mutants two different versions withimproved ΔΔG (S15 and S23) were used either as whole IgG or expressed asscFv on K562 cell surface for functionality assays. A79-scFv is thenfused in frame with truncated human IgG₁-Fc sequence. cDNA representingmutant A79scFv was synthesized de novo as gene string products(Geneart)), then cloned into a plasmid backbone containing SV40 asorigin of replication with promoter element ended with SV40 polyA tail.Antibody was expressed as soluble protein in mammalian cells viadesignated expression plasmid. Secreted antibody was purified fromtissue culture supernatant by affinity purification (Protein A) and thendialyzed to PBS (pH 7.2). Recombinant antibody purity was checked onSDS-PAGE, protein concentration was measured by BCA (Thermoscientific).Aliquots of recombinant antibody was stored frozen in −80° C. until use.

Site directed mutagenesis in 79A CDRs resulted in reformatting a mutantrecombinant antibody S15 (Single chain Affinity optimized novel mutant15) which showed improve affinity and conformation-specific binding topan-specific TCR alpha chain (FIGS. 6A-6C). Gel electrophoresis imageshows homogeneity and purity of recombinant mutant 23 (S23) (FIG. 6D).Additional binding data provided using S15 in FIG. 6E.

Vector design and surface expression of recombinant antibody S15 on K562HLAC-cells—S15 sequences were fused either to truncated CD8transmembrane (tCD8-TM) or truncated human Fc-tCD8-TM domain (with andwithout HA tag (FIG. 7A)) and expressed on K562C-cells along withco-stimulatory ligands C86, CD137L and IL15-15Ra (FIG. 7B). Vectordesign and expression was similar for S23 sequences.

Cell proliferation assay—To assess proliferation of cells afterstimulation through CD3-complex OKT3 or αβ-TCR engagement via mutantscFvs, a CFSE dye dilution method (CellTrace, ThermoFisher Scientific)was used. Live cells were loaded with 5 uM of CFSE dye (as permanufacturer's protocol) and then seeded at a density of 1 millioncells/mL in complete RPMI-1640 media (10% FBS, 1× Glutamax and IL-2;50IU/mL IL-2) on to polystyrene plate (Nunc) coated with either OKT3,S15, S23 alone or in combination with CD28 at 10 μg/mL concentration.Activated cells were sampled on Day 1, 2 and Day 6 and run on flowcytometry for quantification of CFSE diminution. Live CD3+ T cells wereanalyzed for CFSE fluorescence with 488nm excitation on an appropriatefilter on a BD LSR-Fortessa. Data were represented as histogram showingCFSE expression as distinct peak for each successive generation of Tcells (FIGS. 7C and 7D). CFSE loaded PBMC was used as positive control.A commercially available anti-CD28 antibody was used as an experimentalantibody control for T cell simulation.

K562-engineered artificial antigen presenting cells—S15 sequences werefused either to truncated human CD8 transmembrane (tCD8TM) domain ortruncated human Fc-tCD8TM domain along with HA tag (FIG. 7A). cDNArepresenting S15-tCD8TM-HA or S15-tCD8TM-tFc fusion protein wassynthesized as gene string products (Geneart). Using gatewayrecombination method S15 was cloned back into plasmid vector pLV300containing HIV regulatory elements, Ig kappa leader peptide sequencesflanked with gateway cloning sites. 293-METR cells were transfected withplasmids for transient expression, following which viral particles wereharvested from culture and then concentrated on Amicon ultra centrifugefilters (Millipore). K562 cells were transfected to express T-cellco-stimulatory ligands such as CD86, CD137L, IL15-15Ra. Surfaceexpression of introduced ligands was assessed by flow cytometry usingcommercially available antibodies for CD86, CD137L, IL15-15Ra (BD) andHA (Biolegend) or anti-human Fc antibodies (Life Tech) to detect S15fusion protein. To assess efficacy of recombinant antibody to stimulateT cells in absence of co-stimulation, K562 cells were engineered toexpress CD64 and then loaded with whole antibodies as describedpreviously.

Ex vivo propagation of T cells on K562-S15 feeder cells (AaPC)—K562cells were engineered to express S15 or its derivative along with T cellco-stimulatory ligands so that T cell activation is complete ex vivo.K562 cells stably expressing S15 alone or in combination withco-stimulatory ligands or K562-CD64 cells loaded with whole antibodywere used as feeder cells after irradiation (FIG. 7B). PBMC or umbilicalcord blood T cells (UCB) derived mononuclear cells were co-cultured withengineered irradiated K562s at a ratio of 1:1 along with IL-2 (50IU/mL). Cells were stimulated twice at 7-day interval in a K562-PBMCco-culture system. Growth kinetics was monitored by counting live cells(Nexcelom™ trypan blue dye exclusion) (FIGS. 7C-7D). Immunophenotype wascompleted to ascertain the characteristics of expanded T cells (FIG.7E). Emergence of CD3+ CD56^(neg)T cell population was assessed by flowcytometry. Absolute cell number was monitored for estimating cell growthon different aAPCs (K562 cell only, K562 with S15 alone/or S15 withco-stimulatory ligands, K562 with anti-CD3ε mAb (OKT3) alone/or withco-stimulation, or K562-CD64 loaded with 515, S23 or OKT3.Immunophenotyping for ex vivo expanded T cells were performed to assessmarkers related exhaustion and memory phenotypes. (FIGS. 7E and 7F).

Flow Cytometry—BD LSR Fortessa X-20 was used for multiparametricimmunophenotyping analysis of T cells. In brief, healthy donor PBMC orex vivo cultured live T cells were washed by chilled FACS buffercontaining (PBS with 2% FBS), incubated with fluorochrome conjugatedantibodies for 20 min ice in dark. Next, cells were washed twice withFACS buffer and then incubated 5 min at room temp with Fixable viabledye for live dead staining. Gating was performed on live cells andexcluded for monocytes (CD14^(PoS)) and B-cells (CD19^(PoS)) on a dumpchannel. T cells were identified as CD3^(PoS) CD56^(neg) and thenanalyzed for expression of αβ-TCR, markers for cell differentiation(CD25, CD69, CD45RA, and CD45RO), Th Phenotype (CD294 and CD25) andexhaustion (PD1, LAG3, TIM3, BLIMP). Antibody dilution and usages wereperformed as per manufacturer's guidelines. Compensation formulti-parameter analysis was done using calibration beads (eBioscience).Compensated data were analyzed by Flow Jo software V10 and expressed asdot plots. Each experiment was performed for three or more donors. (FIG.7G)

TCR repertoire analysis by high throughput deep sequencing—Activated Tcells' V gene rearrangements were analyzed in-depth by targeting humanTCR-CDR3 region encoded by TCRβ gene (Adaptive Biotechnology's humanTCRβ Deep level assay). The assay involves proprietary multiplex PCRprimers, amplification steps that include a second PCR to incorporatesynthetic immune receptor analogs to avoid amplification bias andaccurate quantification of TCRB repertoire ImmuSEQ™ software (Robins, H.S., Campregher, P. V., Srivastava, S. K., Wacher, A., Turtle, C. J.,Kahsai, O., Riddell, S. R., Warren, E. H. and Carlson, C. S. (2009)Comprehensive assessment of T-cell receptor beta-chain diversity inalphabeta T cells. Blood, 114, 4099-4107.). The results are shown inFIGS. 8A-8B.

Immune repertoire of ex vivo propagated T cells was assessed by nextgeneration deep sequencing of TCRβ gene encoding TCR-CDR3 (hTCRβ deeplevel assay, Adaptive Biotech). Genomic DNA isolated from CD3⁺ T cells(PBMC control) or ex vivo expanded T cells were used for TCR repertoireanalysis. Various APC groups used in the assay were K562 C⁻ (unmodifiedcontrol), K562C⁻ expressing S15 only, K562C⁻ expressing S15 along withco-stimulatory molecules (CD86, CD137L and IL15-15Ra), K562C⁻ loadedwith OKT3 (anti-CD3ε mAb) and co-stimulatory molecules (CD86, CD137L andIL15-15Ra). Amplicons were generated by multiplexing through proprietaryprimer mixture provided in the vendor provided kit. Carlson, C. S.,Emerson, R. O., Sherwood, A. M., Desmarais, C., Chung, M. W., Parsons,J. M., Steen, M. S., LaMadrid-Herrmannsfeldt, M. A., Williamson, D. W.,Livingston, R. J. et al. (2013) Using synthetic templates to design anunbiased multiplex PCR assay. Nature communications, 4, 268. A two-stepamplification process was followed where synthetic immune receptoranalogs were introduced for unbiased estimation of TCRβ repertoire.Robins, H. S., Campregher, P. V., Srivastava, S. K., Wacher, A., Turtle,C. J., Kahsai, O., Riddell, S. R., Warren, E. H. and Carlson, C. S.(2009) Comprehensive assessment of T-cell receptor beta-chain diversityin alphabeta T cells. Blood, 114, 4099-4107. TCR libraries weregenerated on Illumina miSEQ platform and data analyzed by ImmunoSeQanalyzer after a standard QC check (Adaptive Biotech). Immune sequencingdata were obtained from each group of activated T cells as well as fromun-manipulated healthy donor PBMC T cells. All data were compared beforeand after manipulation (primary versus activated T cells). Datarepresenting TCRV gene frequency, CDR3 chain length, paired genefrequency was reported. Top clone distribution was also tracked based onpercentage of sequencing reads accounted for each individual clonewithin sample groups. Scatter plot analysis was provided to estimatefrequency of appearance of clones for each sample group. Clonality scorewith respect to Shannon's entropy, Pearson co-efficient values (r²) werecalculated to ascertain clonal overlap occurring among each group of Tcells activated by different strategies (S15 vs. OKT3 as compared toun-manipulated donor PBMC T cells). (FIG. 8D).

Discussion—The inventors describe an alternate method of T-cellactivation and ex vivo propagation to generate clinical grade T cells.In contrast to CD3 complex activation via CDR3ε-specific mAb OKT3 alongwith super-agonist anti-CD28 mAb, they activated T cells by αβ-TCRcross-linking via an engineered TCR alpha chain specific recombinantantibody fragment tethered on K562-AaPC (activating and propagatingcells).

Single chain affinity optimized novel mutants 15 and 23 (S15 and S23)with high affinity towards αβ-TCR⁺T cells, which were used to activateand propagate primary T cells in culture by cross-linking TCR. Growthkinetics and phenotype of S15-AaPC mediated T cell activation wasparallel to that achieved by CD3ε specific OKT3 mAb stimulation.

High throughput deep sequencing of activated T cells' genomic DNArevealed distinct V-gene rearrangements and preservation of clonalpopulation by TCR-cross linking via S15. The analysis showed neither anyunexpected change in functional V gene rearrangement nor any skewing ofimmune repertoire. αβ⁺ TCR⁺ T-cells generated via TCR cross-linking canbe helpful in situations where preservation of clonal population isdesirable after ex vivo activation and propagation. (see FIGS. 9-11).

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

1-111. (canceled)
 112. An isolated antibody or antigen-binding fragmentthereof that specifically binds to an epitope of T-cell receptor alpha(TCRα) polypeptide, wherein the antibody or antigen-binding fragmentthereof comprises a heavy chain variable region (VH) and a light chainvariable region (VL) wherein the VHcomprises three complementaritydetermining regions (CDRs) HCDR1, HCDR2, and HCDR3, found within theamino acid sequence of any one of SEQ ID NO: 11, 13 or 19 and whereinthe VLcomprises three CDRs, LCDR1, LCDR2 and LCDR3, found within theamino acid sequence of any one of SEQ ID NO: 12, 14, or
 20. 113. Theantibody or antigen-binding fragment thereof according to claim 112,wherein (a) HCDR1 comprises the amino acid sequence of SEQ ID NO: 2; orSEQ ID NO: 15; (b) HCDR2 comprises the amino acid sequence of SEQ ID NO:3; or SEQ ID NO: 16; (c) HCDR3 comprises the amino acid sequence of SEQID NO: 21; SEQ ID NO:23; SEQ ID NO:22 or SEQ ID NO: 17; (d) LCDR1comprises the amino acid sequence of SEQ ID NO: 4; SEQ ID NO: 9; or SEQID NO:18; (e) LCDR2 comprises the amino acid sequence of SEQ ID NO: 5;and (f) LCDR3 comprises the amino acid sequence of SEQ ID NO: 6; or SEQID NO:
 10. 114. The antibody or antigen-binding fragment thereofaccording to claim 112, wherein (a) HCDR1 comprises the amino acidsequence of SEQ ID NO: 15; (b) HCDR2 comprises the amino acid sequenceof SEQ ID NO: 16; (c) HCDR3 comprises the amino acid sequence of SEQ IDNO: 17; (d) LCDR1 comprises the amino acid sequence of SEQ ID NO: 4 orSEQ ID NO: 18; (e) LCDR2 comprises the amino acid sequence of SEQ ID NO:5; and (f) LCDR3 comprises the amino acid sequence of SEQ ID NO:
 6. 115.The antibody or antigen-binding fragment thereof according to claim 112,wherein (a) HCDR1 comprises the amino acid sequence of SEQ ID NO: 15;(b) HCDR2 comprises the amino acid sequence of SEQ ID NO: 16; (c) HCDR3comprises the amino acid sequence of SEQ ID NO: 17; (d) LCDR1 comprisesthe amino acid sequence of SEQ ID NO: 4; (e) LCDR2 comprises the aminoacid sequence of SEQ ID NO: 5; and (f) LCDR3 comprises the amino acidsequence of SEQ ID NO:
 6. 116. The antibody or antigen-binding fragmentthereof according to claim 112, wherein (a) HCDR1 comprises the aminoacid sequence of SEQ ID NO: 15; (b) HCDR2 comprises the amino acidsequence of SEQ ID NO: 16; (c) HCDR3 comprises the amino acid sequenceof SEQ ID NO: 17; (d) LCDR1 comprises the amino acid sequence of SEQ IDNO: 18; (e) LCDR2 comprises the amino acid sequence of SEQ ID NO: 5; and(f) LCDR3 comprises the amino acid sequence of SEQ ID NO:
 6. 117. Theantibody or antigen-binding fragment thereof according to claim 112,wherein the antibody or antigen-binding fragment thereof is human orhumanized.
 118. The antibody or antigen-binding fragment thereofaccording to claim 112, wherein the antigen-binding fragment thereof isa Fab, Fab′, F(ab′)2, scFv, or disulfide-linked Fv (sdFv).
 119. Theantibody or antigen-binding fragment thereof according to claim 112,wherein the antibody is an IgG, IgM or IgA antibody.
 120. The antibodyor antigen-binding fragment thereof according to claim 112, wherein theantibody or antigen-binding fragment thereof is membrane bound.
 121. Theantibody or antigen-binding fragment thereof according to claim 112,wherein the antibody or antigen-binding fragment thereof is conjugatedto a reporter.
 122. The antibody or antigen-binding fragment thereofaccording to claim 112, wherein the antibody or antigen-binding fragmentthereof comprises a heavy chain variable region (VH) comprising theamino acid sequence of SEQ ID NO: 11, and a light chain variable region(VL) comprising the amino acid sequence of SEQ ID NO:
 12. 123. Theantibody or antigen-binding fragment thereof according to claim 112,wherein the antibody or antigen-binding fragment thereof comprises a VHcomprising the amino acid sequence of SEQ ID NO: 11, and a VL comprisingthe amino acid sequence of SEQ ID NO:
 14. 124. The antibody orantigen-binding fragment thereof according to claim 112, wherein theantibody or antigen-binding fragment thereof comprises a VH comprisingthe amino acid sequence of SEQ ID NO: 13, and a VL comprising the aminoacid sequence of SEQ ID NO:
 12. 125. The antibody or antigen-bindingfragment thereof according to claim 112, wherein the antibodyantigen-binding fragment thereof comprises a VH comprising the aminoacid sequence of SEQ ID NO: 19, and a VL comprising the amino acidsequence of SEQ ID NO:
 20. 126. The antibody or antigen-binding fragmentthereof according to claim 112, wherein the epitope of the TCRαpolypeptide comprises GSTLRG (SEQ ID NO:1).
 127. The antibody orantigen-binding fragment thereof according to claim 112, furthercomprising a transmembrane domain.
 128. An isolated polynucleotidecomprising a nucleic acid sequence encoding the antibody or antigenbinding fragment thereof according to claim
 112. 129. An isolated hostcell comprising one or more polynucleotides according to claim
 128. 130.A method for selecting a cell comprising a TCRα comprising: (i)contacting the cell with an antibody or antigen-binding fragment thereofaccording to claim 112; and (ii) selecting a cell comprising the TCRα.13. A method for expanding and/or activating T-cells comprisingcontacting the T-cells with artificial antigen presenting cells (aAPCs)in the presence of an antibody or antigen binding fragment thereofaccording to claim
 112. 132. A method of treating an autoimmune diseaseor a T-cell leukemia in a subject in need of treatment, comprisingadministering an antibody or antigen-binding fragment thereof accordingto claim 112.